Optimized cross-species specific bispecific single chain antibody contructs

ABSTRACT

The present invention provides to a bispecific single chain antibody construct binding to a target cell surface antigen via a first binding domain and to the T cell surface antigen CD3 via a second binding domain, wherein serum albumin is fused to the C-terminus of the antibody construct. Moreover, the invention provides a polynucleotide encoding the antibody construct, a vector comprising said polynucleotide and a host cell transformed or transfected with said vector. Furthermore, the invention provides a process for the production of the antibody construct of the invention, a medical use of said antibody construct and a kit comprising said antibody construct.

FIELD OF THE INVENTION

The present invention relates to a bispecific single chain antibodyconstruct binding to a target cell surface antigen via a first bindingdomain and to the T cell surface antigen CD3 via a second bindingdomain, wherein serum albumin is fused to the C-terminus of the antibodyconstruct. Moreover, the invention provides a polynucleotide encodingthe antibody construct, a vector comprising said polynucleotide and ahost cell transformed or transfected with said vector. Furthermore, theinvention provides a process for the production of the antibodyconstruct of the invention, a medical use of said antibody construct anda kit comprising said antibody construct.

BACKGROUND OF THE INVENTION

Bispecific molecules such as BiTE® (bispecific T cell engager)antibodies are recombinant protein constructs made from two flexiblylinked antibody derived binding domains. One binding domain of BiTE®antibodies is specific for a selected tumor-associated surface antigenon target cells; the second binding domain is specific for CD3, asubunit of the T cell receptor complex on T cells. By their particulardesign BiTE® antibodies are uniquely suited to transiently connect Tcells with target cells and, at the same time, potently activate theinherent cytolytic potential of T cells against target cells. Animportant further development of the first generation of BiTE® molecules(see WO 99/54440 and WO 2005/040220) developed into the clinic as AMG103 and AMG 110 was the provision of bispecific antibody constructsbinding to a context independent epitope at the N-terminus of the CD3Echain (WO 2008/119567). BiTE® antibodies binding to this elected epitopedid not only show cross-species specificity for human and Callithrixjacchus, Saguinus oedipus or Saimiri sciureus CD3ε chain, but also, dueto using this epitope instead of previously described epitopes for CD3binders in bispecific T cell engaging molecules, do not activate T cellsto the same degree as observed for the previous generation of T cellengaging antibodies. This difference in T cell activation was connectedwith less or reduced T cell redistribution in patients, which wasidentified as a risk for side effects.

DEFINITIONS

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within ±20%,preferably within ±15%, more preferably within ±10%, and most preferablywithin ±5% of a given value or range.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

DETAILED DESCRIPTION OF THE INVENTION

For bispecific T cell engaging molecules comprising as their T cellbinding entity a binding domain specific for the human and Callithrixjacchus, Saguinus oedipus or Saimiri sciureus CD3ε chain, wherein theepitope is part of an amino acid sequence comprised in the groupconsisting of SEQ ID NOs: 7, 8, 9, and 10 and comprises at least theamino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 1), it was observedthat those molecules when used in extremely high concentration showed Tcell cytotoxicity, independent of the target specificity of the secondbinding domain and even in the absence of target cells. Although thosehigh concentrations of bispecific T cell engaging molecules are notrelevant for every combination of disease and target in a treatmentusing the mode of action of T cell engaging molecules in case of acontinuous i.v. administration, such high concentration issues maybecome relevant for specific administration routes or in combinationwith specific target settings and required compound concentrations.

Thus, the problem underlying the present invention was to providebispecific single chain antibody constructs using a T cell bindingdomain of the latest generation, which avoid the T cell cytotoxicityeven in high compound concentrations.

This problem was solved by providing a bispecific single chain antibodyconstruct binding to a target cell surface antigen via a first bindingdomain and the T cell surface antigen CD3 via a second binding domain,wherein:

-   -   the second binding domain binds to an epitope of human and        Callithrix jacchus, Saguinus oedipus or Saimiri sciureus CD3ε        chain, wherein the epitope is part of an amino acid sequence        comprised in the group consisting of SEQ ID NO: 7 (human), SEQ        ID NO: 8 (Callithrix jacchus), SEQ ID NO: 9 (Saguinus oedipus),        and SEQ ID NO: 10 (Saimiri sciureus) and comprises at least the        amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 1). and    -   a serum albumin is fused to the C-terminus of the antibody        construct;        wherein the bispecific single chain antibody construct does not        have an amino acid sequence as depicted in SEQ ID NOs: 2 and 3.

Without the intention to be bound by theory, the activation of T cellsin the presence of a high concentration of the T cell engagingbispecific antibody constructs and in the absence of target cells isexplained by dimerization or multimerization of the antibody constuctsvia the CD3 binding domain. Such di- or multimerization is stericallyimpaired by the fusion of an albumin or variant thereof to the Cterminus of the antibody construct, while maintaining thecharacteristics of the antibody construct for its T cell engaging modeof action. Those high concentrations of the T cell engaging bispecificantibody constructs in a patient are those concentrations in a specificcompartment such as the serum. The fusion of the albumin to theC-terminus of the T cell engaging antibody construct prevents theactivation of T cells in the absence of target cells up to aconcentration of 2 mg/ml, at least up to 1 mg/ml, more preferably up to500 ng/ml.

Callithrix jacchus and Saguinus oedipus are both new world primatebelonging to the family of Callitrichidae, while Saimiri sciureus is anew world primate belonging to the family of Cebidae.

Serum albumin is a protein physiologically produced by the liver; itoccurs dissolved in blood plasma and is the most abundant blood proteinin mammals. Albumin is essential for maintaining the oncotic pressureneeded for proper distribution of body fluids between blood vessels andbody tissues. It also acts as a plasma carrier by non-specificallybinding several hydrophobic steroid hormones and as a transport proteinfor hemin and fatty acids. The term “serum albumin” respectively thehuman variant thereof (“human albumin”) defines in the context of theinvented proteins either the parental human serum albumin protein(sequence as described in SEQ ID NO: 4) or any variant (e.g. such asalbumin protein as depicted in SEQ ID NOs: 5-12 or 608 to 628) orfragment thereof preferably expressed as genetic fusion proteins and bychemical crosslinking etc. at least with one therapeutic protein.Variants comprising single or multiple mutations or fragments of albuminprovide improved properties such as affinities to FcRn receptor andextended plasma half-life compared to its parent or reference. Apreferred group of serum albumin sequences in connection with thisinvention is selected from a group consisting of SEQ ID NOs: 4, 6, 7, 8,9, 10, 11, 12, 608, 609, 611, 612, 613, 614, 615, 616, 617, 618, 619,621, 622, 623, 624, 625, 626, 627, and 628.

The term “antibody construct” refers to a molecule in which thestructure and/or function is/are based on the structure and/or functionof an antibody, e.g. of a full-length or whole immunoglobulin molecule.An antibody construct is hence capable of binding to its specific targetor antigen. Furthermore, an antibody construct according to theinvention comprises the minimum structural requirements of an antibodywhich allow for the target binding. This minimum requirement may e.g. bedefined by the presence of at least the three light chain CDRs (i.e.CDR1, CDR2 and CDR3 of the VL region) and/or the three heavy chain CDRs(i.e. CDR1, CDR2 and CDR3 of the VH region). The antibodies on which theconstructs according to the invention are based include for examplemonoclonal, recombinant, chimeric, deimmunized, humanized and humanantibodies.

Within the definition of “antibody constructs” according to theinvention are full-length or whole antibodies including camelidantibodies and other immunoglobulin antibodies generated bybiotechnological or protein engineering methods or processes. Thesefull-length antibodies may be for example monoclonal, recombinant,chimeric, deimmunized, humanized and human antibodies. Also within thedefinition of “antibody constructs” are fragments of full-lengthantibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab′, F(ab′)2 or“r IgG” (“half antibody”). Antibody constructs according to theinvention may also be modified fragments of antibodies, also calledantibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc,scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies,tandem diabodies (Tandab′s), tandem di-scFv, tandem tri-scFv,“minibodies” exemplified by a structure which is as follows:(VH-VL-CH3)2, (scFv-CH3)2 or (scFv-CH3-scFv)2, multibodies such astriabodies or tetrabodies, and single domain antibodies such asnanobodies or single variable domain antibodies comprising merely onevariable domain, which might be VHH, VH or VL, that specifically bind anantigen or epitope independently of other V regions or domains.

Furthermore, the definition of the term “antibody constructs” includesmonovalent, bivalent and polyvalent/multivalent constructs and, thus,monospecific constructs, specifically binding to only one antigenicstructure, as well as bispecific and polyspecific/multispecificconstructs, which specifically bind more than one antigenic structure,e.g. two, three or more, through distinct binding domains. Moreover, thedefinition of the term “antibody constructs” includes moleculesconsisting of only one polypeptide chain as well as molecules consistingof more than one polypeptide chain, which chains can be either identical(homodimers, homotrimers or homo oligomers) or different (heterodimer,heterotrimer or heterooligomer). Examples for the above identifiedantibodies and variants or derivatives thereof are described inter aliain Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988)and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermannand Dübel, Antibody Engineering, Springer, 2nd ed. 2010 and Little,Recombinant Antibodies for Immunotherapy, Cambridge University Press2009.

The antibody constructs of the present invention are preferably “invitro generated antibody constructs”. This term refers to an antibodyconstruct according to the above definition where all or part of thevariable region (e.g., at least one CDR) is generated in a non-immunecell selection, e.g., an in vitro phage display, protein chip or anyother method in which candidate sequences can be tested for theirability to bind to an antigen. This term thus preferably excludessequences generated solely by genomic rearrangement in an immune cell inan animal. A “recombinant antibody” is an antibody made through the useof recombinant DNA technology or genetic engineering.

The present invention is directed to “single chain antibody constructs”.Accordingly, those single chain antibody constructs only include thoseembodiments of the above described antibody constructs, which consist ofa single peptide chain.

The term “monoclonal antibody” (mAb) or monoclonal antibody construct asused herein refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations and/or post-translation modifications (e.g.,isomerizations, amidations) that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site or determinant on the antigen, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (orepitopes). In addition to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,hence uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod.

For the preparation of monoclonal antibodies, any technique providingantibodies produced by continuous cell line cultures can be used. Forexample, monoclonal antibodies to be used may be made by the hybridomamethod first described by Koehler et al., Nature, 256: 495 (1975), ormay be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). Examples for further techniques to produce human monoclonalantibodies include the trioma technique, the human B-cell hybridomatechnique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985), 77-96).

Hybridomas can then be screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance(BIACORE™) analysis, to identify one or more hybridomas that produce anantibody that specifically binds with a specified antigen. Any form ofthe relevant antigen may be used as the immunogen, e.g., recombinantantigen, naturally occurring forms, any variants or fragments thereof,as well as an antigenic peptide thereof. Surface plasmon resonance asemployed in the BIAcore system can be used to increase the efficiency ofphage antibodies which bind to an epitope of a target antigen, such asthe target cell surface antigen or CD3 epsilon (Schier, Human AntibodiesHybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995),7-13).

Another exemplary method of making monoclonal antibodies includesscreening protein expression libraries, e.g., phage display or ribosomedisplay libraries. Phage display is described, for example, in Ladner etal., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317,Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol.Biol., 222: 581-597 (1991).

In addition to the use of display libraries, the relevant antigen can beused to immunize a non-human animal, e.g., a rodent (such as a mouse,hamster, rabbit or rat). In one embodiment, the non-human animalincludes at least a part of a human immunoglobulin gene. For example, itis possible to engineer mouse strains deficient in mouse antibodyproduction with large fragments of the human Ig (immunoglobulin) loci.Using the hybridoma technology, antigen-specific monoclonal antibodiesderived from the genes with the desired specificity may be produced andselected. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics7:13-21, US 2003-0070185, WO 96/34096, and WO96/33735.

A monoclonal antibody can also be obtained from a non-human animal, andthen modified, e.g., humanized, deimmunized, rendered chimeric etc.,using recombinant DNA techniques known in the art. Examples of modifiedantibody constructs include humanized variants of non-human antibodies,“affinity matured” antibodies (see, e.g. Hawkins et al. J. Mol. Biol.254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837(1991)) and antibody mutants with altered effector function(s) (see,e.g., U.S. Pat. No. 5,648,260, Kontermann and Dübel (2010), loc. cit.and Little (2009), loc. cit.).

In immunology, affinity maturation is the process by which B cellsproduce antibodies with increased affinity for antigen during the courseof an immune response. With repeated exposures to the same antigen, ahost will produce antibodies of successively greater affinities. Likethe natural prototype, the in vitro affinity maturation is based on theprinciples of mutation and selection. The in vitro affinity maturationhas successfully been used to optimize antibodies, antibody constructs,and antibody fragments. Random mutations inside the CDRs are introducedusing radiation, chemical mutagens or error-prone PCR. In addition, thegenetical diversity can be increased by chain shuffling. Two or threerounds of mutation and selection using display methods like phagedisplay usually results in antibody fragments with affinities in the lownanomolar range.

A preferred type of an amino acid substitutional variation of theantibody constructs involves substituting one or more hypervariableregion residues of a parent antibody (e. g. a humanized or humanantibody). Generally, the resulting variant(s) selected for furtherdevelopment will have improved biological properties relative to theparent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e. g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e. g. binding affinity) as herein disclosed. In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the bindingdomain and, e.g., human the target cell surface antigen. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

The monoclonal antibodies and antibody constructs of the presentinvention specifically include “chimeric” antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816, 567; Morrison et al., Proc.Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primitized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc.) and human constant region sequences. Avariety of approaches for making chimeric antibodies have beendescribed. See e.g., Morrison et al., Proc. Natl. Acad. ScL U.S.A.81:6851 , 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al.,U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchiet al., EP 0171496; EP 0173494; and GB 2177096.

An antibody, antibody construct or antibody fragment may also bemodified by specific deletion of human T cell epitopes (a method called“deimmunization”) by the methods disclosed in WO 98/52976 and WO00/34317. Briefly, the heavy and light chain variable domains of anantibody can be analyzed for peptides that bind to MHC class II; thesepeptides represent potential T cell epitopes (as defined in WO 98/52976and WO 00/34317). For detection of potential T cell epitopes, a computermodeling approach termed “peptide threading” can be applied, and inaddition a database of human MHC class II binding peptides can besearched for motifs present in the VH and VL sequences, as described inWO 98/52976 and WO 00/34317. These motifs bind to any of the 18 majorMHC class II DR allotypes, and thus constitute potential T cellepitopes. Potential T cell epitopes detected can be eliminated bysubstituting small numbers of amino acid residues in the variabledomains, or preferably, by single amino acid substitutions. Typically,conservative substitutions are made. Often, but not exclusively, anamino acid common to a position in human germline antibody sequences maybe used. Human germline sequences are disclosed e.g. in Tomlinson, etal. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995) Immunol.Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The V BASE directory provides a comprehensive directory ofhuman immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). Thesesequences can be used as a source of human sequence, e.g., for frameworkregions and CDRs. Consensus human framework regions can also be used,for example as described in U.S. Pat. No. 6,300,064.

“Humanized” antibodies, antibody constructs or fragments thereof (suchas Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences ofantibodies) are antibodies or immunoglobulins of mostly human sequences,which contain (a) minimal sequence(s) derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region (also CDR) of the recipient are replaced byresidues from a hypervariable region of a non-human (e.g., rodent)species (donor antibody) such as mouse, rat, hamster or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, “humanized antibodies”as used herein may also comprise residues which are found neither in therecipient antibody nor the donor antibody. These modifications are madeto further refine and optimize antibody performance. The humanizedantibody may also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321: 522-525 (1986);Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op.Struct. Biol., 2: 593-596 (1992).

Humanized antibodies or fragments thereof can be generated by replacingsequences of the Fv variable domain that are not directly involved inantigen binding with equivalent sequences from human Fv variabledomains. Exemplary methods for generating humanized antibodies orfragments thereof are provided by Morrison (1985) Science 229:1202-1207;by Oi et al. (1986) BioTechniques 4:214; and by U.S. Pat. No. 5,585,089;U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No.5,859,205; and U.S. Pat. No. 6,407,213. Those methods include isolating,manipulating, and expressing the nucleic acid sequences that encode allor part of immunoglobulin Fv variable domains from at least one of aheavy or light chain. Such nucleic acids may be obtained from ahybridoma producing an antibody against a predetermined target, asdescribed above, as well as from other sources. The recombinant DNAencoding the humanized antibody molecule can then be cloned into anappropriate expression vector.

Humanized antibodies may also be produced using transgenic animals suchas mice that express human heavy and light chain genes, but areincapable of expressing the endogenous mouse immunoglobulin heavy andlight chain genes. Winter describes an exemplary CDR grafting methodthat may be used to prepare the humanized antibodies described herein(U.S. Pat. No. 5,225,539). All of the CDRs of a particular humanantibody may be replaced with at least a portion of a non-human CDR, oronly some of the CDRs may be replaced with non-human CDRs. It is onlynecessary to replace the number of CDRs required for binding of thehumanized antibody to a predetermined antigen.

A humanized antibody can be optimized by the introduction ofconservative substitutions, consensus sequence substitutions, germlinesubstitutions and/or back mutations. Such altered immunoglobulinmolecules can be made by any of several techniques known in the art,(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983;Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth.Enzymol., 92: 3-16, 1982, and EP 239 400.

The term “human antibody”, “human antibody construct” and “human bindingdomain” includes antibodies, antibody constructs and binding domainshaving antibody regions such as variable and constant regions or domainswhich correspond substantially to human germline immunoglobulinsequences known in the art, including, for example, those described byKabat et al. (1991) (loc. cit.). The human antibodies, antibodyconstructs or binding domains of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs, and inparticular, in CDR3. The human antibodies, antibody constructs orbinding domains can have at least one, two, three, four, five, or morepositions replaced with an amino acid residue that is not encoded by thehuman germline immunoglobulin sequence. The definition of humanantibodies, antibody constructs and binding domains as used herein alsocontemplates fully human antibodies, which include only non-artificiallyand/or genetically altered human sequences of antibodies as those can bederived by using technologies or systems such as the Xenomouse.

In some embodiments, the antibody constructs of the invention are“isolated” or “substantially pure” antibody constructs. “Isolated” or“substantially pure” when used to describe the antibody constructdisclosed herein means an antibody construct that has been identified,separated and/or recovered from a component of its productionenvironment. Preferably, the antibody construct is free or substantiallyfree of association with all other components from its productionenvironment. Contaminant components of its production environment, suchas that resulting from recombinant transfected cells, are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. The antibody constructs may e.g.constitute at least about 5%, or at least about 50% by weight of thetotal protein in a given sample. It is understood that the isolatedprotein may constitute from 5% to 99.9% by weight of the total proteincontent, depending on the circumstances. The polypeptide may be made ata significantly higher concentration through the use of an induciblepromoter or high expression promoter, such that it is made at increasedconcentration levels. The definition includes the production of anantibody construct in a wide variety of organisms and/or host cells thatare known in the art. In preferred embodiments, the antibody constructwill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Ordinarily, however, an isolated antibody construct willbe prepared by at least one purification step.

The term “binding domain” characterizes in connection with the presentinvention a domain which (specifically) binds to/interactswith/recognizes a given target epitope or a given target site on thetarget molecules (antigens) and CD3, respectively. The structure andfunction of the first binding domain (recognizing the target cellsurface antigen), and preferably also the structure and/or function ofthe second binding domain (CD3), is/are based on the structure and/orfunction of an antibody, e.g. of a full-length or whole immunoglobulinmolecule. According to the invention, the first binding domain ischaracterized by the presence of three light chain CDRs (i.e. CDR1, CDR2and CDR3 of the VL region) and three heavy chain CDRs (i.e. CDR1, CDR2and CDR3 of the VH region). The second binding domain preferably alsocomprises the minimum structural requirements of an antibody which allowfor the target binding. More preferably, the second binding domaincomprises at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 ofthe VL region) and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3of the VH region). It is envisaged that the first and/or second bindingdomain is produced by or obtainable by phage-display or libraryscreening methods rather than by grafting CDR sequences from apre-existing (monoclonal) antibody into a scaffold.

According to the present invention, binding domains are preferably inthe form of polypeptides. Such polypeptides may include proteinaceousparts and non-proteinaceous parts (e.g. chemical linkers or chemicalcross-linking agents such as glutaraldehyde). Proteins (includingfragments thereof, preferably biologically active fragments, andpeptides, usually having less than 30 amino acids) comprise two or moreamino acids coupled to each other via a covalent peptide bond (resultingin a chain of amino acids). The term “polypeptide” as used hereindescribes a group of molecules, which usually consist of more than 30amino acids. Polypeptides may further form multimers such as dimers,trimers and higher oligomers, i.e. consisting of more than onepolypeptide molecule. Polypeptide molecules forming such dimers, trimersetc. may be identical or non-identical. The corresponding higher orderstructures of such multimers are, consequently, termed homo- orheterodimers, homo- or heterotrimers etc. An example for ahereteromultimer is an antibody molecule, which, in its naturallyoccurring form, consists of two identical light polypeptide chains andtwo identical heavy polypeptide chains. The terms “peptide”,“polypeptide” and “protein” also refer to naturally modifiedpeptides/polypeptides/proteins wherein the modification is effected e.g.by post-translational modifications like glycosylation, acetylation,phosphorylation and the like. A “peptide”, “polypeptide” or “protein”when referred to herein may also be chemically modified such aspegylated. Such modifications are well known in the art and describedherein below.

As mentioned above, a binding domain may typically comprise an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH); however, it does not have to comprise both. Fd fragments,for example, have two VH regions and often retain some antigen-bindingfunction of the intact antigen-binding domain. Examples of (modified)antigen-binding antibody fragments include (1) a Fab fragment, amonovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab′)2fragment, a bivalent fragment having two Fab fragments linked by adisulfide bridge at the hinge region; (3) an Fd fragment having the twoVH and CH1 domains; (4) an Fv fragment having the VL and VH domains of asingle arm of an antibody, (5) a dAb fragment (Ward et al., (1989)Nature 341 :544-546), which has a VH domain; (6) an isolatedcomplementarity determining region (CDR), and (7) a single chain Fv(scFv), the latter being preferred (for example, derived from anscFv-library).

Antibodies and antibody constructs comprising at least one human bindingdomain avoid some of the problems associated with antibodies or antibodyconstructs that possess non-human such as rodent (e.g. murine, rat,hamster or rabbit) variable and/or constant regions. The presence ofsuch rodent derived proteins can lead to the rapid clearance of theantibodies or antibody constructs or can lead to the generation of animmune response against the antibody or antibody construct by a patient.In order to avoid the use of rodent derived antibodies or antibodyconstructs, human or fully human antibodies/antibody constructs can begenerated through the introduction of human antibody function into arodent so that the rodent produces fully human antibodies.

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the use of such technology for substitution ofmouse loci with their human equivalents could provide unique insightsinto the expression and regulation of human gene products duringdevelopment, their communication with other systems, and theirinvolvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (mAbs)—an important milestone towards fulfilling the promiseof antibody therapy in human disease. Fully human antibodies or antibodyconstructs are expected to minimize the immunogenic and allergicresponses intrinsic to mouse or mouse-derivatized mAbs and thus toincrease the efficacy and safety of the administered antibodies/antibodyconstructs. The use of fully human antibodies or antibody constructs canbe expected to provide a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated compoundadministrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human mAbs with thedesired specificity could be readily produced and selected. This generalstrategy was demonstrated in connection with the generation of the firstXenoMouse mouse strains (see Green et al. Nature Genetics 7:13-21(1994)). The XenoMouse strains were engineered with yeast artificialchromosomes (YACs) containing 245 kb and 190 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus, respectively, which contained core variable and constantregion sequences. The human Ig containing YACs proved to be compatiblewith the mouse system for both rearrangement and expression ofantibodies and were capable of substituting for the inactivated mouse Iggenes. This was demonstrated by their ability to induce B celldevelopment, to produce an adult-like human repertoire of fully humanantibodies, and to generate antigen-specific human mAbs. These resultsalso suggested that introduction of larger portions of the human Ig locicontaining greater numbers of V genes, additional regulatory elements,and human Ig constant regions might recapitulate substantially the fullrepertoire that is characteristic of the human humoral response toinfection and immunization. The work of Green et al. was recentlyextended to the introduction of greater than approximately 80% of thehuman antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci andkappa light chain loci, respectively. See Mendez et al. Nature Genetics15:146-156 (1997) and U.S. patent application Ser. No. 08/759,620.

The production of the XenoMouse mice is further discussed and delineatedin U.S. patent applications Ser. No. 07/466,008, Ser. No. 07/610,515,Ser. No. 07/919,297, Ser. No. 07/922,649, Ser. No. 08/031,801, Ser. No.08/112,848, Ser. No. 08/234,145, Ser. No. 08/376,279, Ser. No.08/430,938, Ser. No. 08/464,584, Ser. No. 08/464,582, Ser. No.08/463,191, Ser. No. 08/462,837, Ser. No. 08/486,853, Ser. No.08/486,857, Ser. No. 08/486,859, Ser. No. 08/462,513, Ser. No.08/724,752, and Ser. No. 08/759,620; and U.S. Pat. Nos. 6,162,963,6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos.3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also Mendez et al.Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.188:483-495 (1998), EP 0 463 151 B1, WO 94/02602, WO 96/34096, WO98/24893, WO 00/76310, and WO 03/47336.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more VH genes, one ormore DH genes, one or more JH genes, a mu constant region, and a secondconstant region (preferably a gamma constant region) are formed into aconstruct for insertion into an animal. This approach is described inU.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806,5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650,5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to Lonberg and Kay,U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns, U.S.Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns et al., and U.S.Pat. No. 5,643,763 to Choi and Dunn, and GenPharm International U.S.patent application Ser. No. 07/574,748, Ser. No. 07/575,962, Ser. No.07/810,279, Ser. No. 07/853,408, Ser. No. 07/904,068, Ser. No.07/990,860, Ser. No. 08/053,131, Ser. No. 08/096,762, Ser. No.08/155,301, Ser. No. 08/161,739, Ser. No. 08/165,699, Ser. No.08/209,741. See also EP 0 546 073 B1, WO 92/03918, WO 92/22645, WO92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175. Seefurther Taylor et al. (1992), Chen et al. (1993), Tuaillon et al.(1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994),and Tuaillon et al. (1995), Fishwild et al. (1996).

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961. Xenerex Biosciences is developinga technology for the potential generation of human antibodies. In thistechnology, SCID mice are reconstituted with human lymphatic cells,e.g., B and/or T cells. Mice are then immunized with an antigen and cangenerate an immune response against the antigen. See U.S. Pat. Nos.5,476,996; 5,698,767; and 5,958,765.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. It is howeverexpected that certain human anti-chimeric antibody (HACA) responses willbe observed, particularly in chronic or multi-dose utilizations of theantibody. Thus, it would be desirable to provide antibody constructscomprising a fully human binding domain against the target cell surfaceantigen and a fully human binding domain against CD3 in order to vitiateconcerns and/or effects of HAMA or HACA response.

The terms “(specifically) binds to”, (specifically) recognizes”, “is(specifically) directed to”, and “(specifically) reacts with” mean inaccordance with this invention that a binding domain interacts orspecifically interacts with one or more, preferably at least two, morepreferably at least three and most preferably at least four amino acidsof an epitope located on the target protein or antigen (the target cellsurface antigen/CD3).

The term “epitope” refers to a site on an antigen to which a bindingdomain, such as an antibody or immunoglobulin or derivative or fragmentof an antibody or of an immunoglobulin, specifically binds. An “epitope”is antigenic and thus the term epitope is sometimes also referred toherein as “antigenic structure” or “antigenic determinant”. Thus, thebinding domain is an “antigen interaction site”. Saidbinding/interaction is also understood to define a “specificrecognition”.

“Epitopes” can be formed both by contiguous amino acids ornon-contiguous amino acids juxtaposed by tertiary folding of a protein.A “linear epitope” is an epitope where an amino acid primary sequencecomprises the recognized epitope. A linear epitope typically includes atleast 3 or at least 4, and more usually, at least 5 or at least 6 or atleast 7, for example, about 8 to about 10 amino acids in a uniquesequence.

A “conformational epitope”, in contrast to a linear epitope, is anepitope wherein the primary sequence of the amino acids comprising theepitope is not the sole defining component of the epitope recognized(e.g., an epitope wherein the primary sequence of amino acids is notnecessarily recognized by the binding domain). Typically aconformational epitope comprises an increased number of amino acidsrelative to a linear epitope. With regard to recognition ofconformational epitopes, the binding domain recognizes athree-dimensional structure of the antigen, preferably a peptide orprotein or fragment thereof (in the context of the present invention,the antigen for one of the binding domains is comprised within the thetarget cell surface antigen protein). For example, when a proteinmolecule folds to form a three-dimensional structure, certain aminoacids and/or the polypeptide backbone forming the conformational epitopebecome juxtaposed enabling the antibody to recognize the epitope.Methods of determining the conformation of epitopes include, but are notlimited to, x-ray crystallography, two-dimensional nuclear magneticresonance (2D-NMR) spectroscopy and site-directed spin labelling andelectron paramagnetic resonance (EPR) spectroscopy.

The interaction between the binding domain and the epitope or epitopecluster implies that a binding domain exhibits appreciable affinity forthe epitope or epitope cluster on a particular protein or antigen (here:the target cell surface antigen and CD3, respectively) and, generally,does not exhibit significant reactivity with proteins or antigens otherthan the target cell surface antigen or CD3. “Appreciable affinity”includes binding with an affinity of about 10⁻⁶ M (KD) or stronger.Preferably, binding is considered specific when the binding affinity isabout 10⁻¹² to 10⁻⁸ M, 10⁻¹² to 10⁻⁹ M, 10⁻¹² to 10⁻¹⁰ M, 10⁻¹¹ to 10⁻⁸M, preferably of about 10⁻¹¹ to 10⁻⁹ M. Whether a binding domainspecifically reacts with or binds to a target can be tested readily by,inter alia, comparing the reaction of said binding domain with a targetprotein or antigen with the reaction of said binding domain withproteins or antigens other than the target cell surface antigen or CD3.Preferably, a binding domain of the invention does not essentially orsubstantially bind to proteins or antigens other than the target cellsurface antigen or CD3 (i.e., the first binding domain is not capable ofbinding to proteins other than the target cell surface antigen and thesecond binding domain is not capable of binding to proteins other thanCD3).

The term “does not essentially/substantially bind” or “is not capable ofbinding” means that a binding domain of the present invention does notbind a protein or antigen other than the target cell surface antigen orCD3, i.e., does not show reactivity of more than 30%, preferably notmore than 20%, more preferably not more than 10%, particularlypreferably not more than 9%, 8%, 7%, 6% or 5% with proteins or antigensother than the target cell surface antigen or CD3, whereby binding tothe target cell surface antigen or CD3, respectively, is set to be 100%.

Specific binding is believed to be effected by specific motifs in theamino acid sequence of the binding domain and the antigen. Thus, bindingis achieved as a result of their primary, secondary and/or tertiarystructure as well as the result of secondary modifications of saidstructures. The specific interaction of the antigen-interaction-sitewith its specific antigen may result in a simple binding of said site tothe antigen. Moreover, the specific interaction of theantigen-interaction-site with its specific antigen may alternatively oradditionally result in the initiation of a signal, e.g. due to theinduction of a change of the conformation of the antigen, anoligomerization of the antigen, etc.

The term “variable” refers to the portions of the antibody orimmunoglobulin domains that exhibit variability in their sequence andthat are involved in determining the specificity and binding affinity ofa particular antibody (i.e., the “variable domain(s)”). The pairing of avariable heavy chain (VH) and a variable light chain (VL) together formsa single antigen-binding site. The CH domain most proximal to VH isdesignated as CH1. Each light (L) chain is linked to a heavy (H) chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype.

Variability is not evenly distributed throughout the variable domains ofantibodies; it is concentrated in sub-domains of each of the heavy andlight chain variable regions. These sub-domains are called“hypervariable regions” or “complementarity determining regions” (CDRs).The more conserved (i.e., non-hypervariable) portions of the variabledomains are called the “framework” regions (FRM) and provide a scaffoldfor the six CDRs in three dimensional space to form an antigen-bindingsurface. The variable domains of naturally occurring heavy and lightchains each comprise four FRM regions (FR1, FR2, FR3, and FR4), largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRM and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site (see Kabat et al., loc. cit.). The constant domainsare not directly involved in antigen binding, but exhibit variouseffector functions, such as, for example, antibody-dependent,cell-mediated cytotoxicity and complement activation.

The terms “CDR”, and its plural “CDRs”, refer to the complementaritydetermining region of which three make up the binding character of alight chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three makeup the binding character of a heavy chain variable region (CDR-H1,CDR-H2 and CDR-H3). CDRs contain most of the residues responsible forspecific interactions of the antibody with the antigen and hencecontribute to the functional activity of an antibody molecule: they arethe main determinants of antigen specificity.

The exact definitional CDR boundaries and lengths are subject todifferent classification and numbering systems. CDRs may therefore bereferred to by Kabat, Chothia, contact or any other boundarydefinitions, including the numbering system described herein. Despitediffering boundaries, each of these systems has some degree of overlapin what constitutes the so called “hypervariable regions” within thevariable sequences. CDR definitions according to these systems maytherefore differ in length and boundary areas with respect to theadjacent framework region. See for example Kabat (an approach based oncross-species sequence variability), Chothia (an approach based oncrystallographic studies of antigen-antibody complexes), and/orMacCallum (Kabat et al., loc. cit.; Chothia et al., J. Mol. Biol, 1987,196: 901-917; and MacCallum et al., J. Mol. Biol, 1996, 262: 732). Stillanother standard for characterizing the antigen binding site is the AbMdefinition used by Oxford Molecular's AbM antibody modeling software.See, e.g., Protein Sequence and Structure Analysis of Antibody VariableDomains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg). To the extent that tworesidue identification techniques define regions of overlapping, but notidentical regions, they can be combined to define a hybrid CDR. However,the numbering in accordance with the so-called Kabat system ispreferred.

Typically, CDRs form a loop structure that can be classified as acanonical structure. The term “canonical structure” refers to the mainchain conformation that is adopted by the antigen binding (CDR) loops.From comparative structural studies, it has been found that five of thesix antigen binding loops have only a limited repertoire of availableconformations. Each canonical structure can be characterized by thetorsion angles of the polypeptide backbone. Correspondent loops betweenantibodies may, therefore, have very similar three dimensionalstructures, despite high amino acid sequence variability in most partsof the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothiaet al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996,263: 800). Furthermore, there is a relationship between the adopted loopstructure and the amino acid sequences surrounding it. The conformationof a particular canonical class is determined by the length of the loopand the amino acid residues residing at key positions within the loop,as well as within the conserved framework (Le., outside of the loop).Assignment to a particular canonical class can therefore be made basedon the presence of these key amino acid residues.

The term “canonical structure” may also include considerations as to thelinear sequence of the antibody, for example, as catalogued by Kabat(Kabat et al., loc. cit.). The Kabat numbering scheme (system) is awidely adopted standard for numbering the amino acid residues of anantibody variable domain in a consistent manner and is the preferredscheme applied in the present invention as also mentioned elsewhereherein. Additional structural considerations can also be used todetermine the canonical structure of an antibody. For example, thosedifferences not fully reflected by Kabat numbering can be described bythe numbering system of Chothia et al and/or revealed by othertechniques, for example, crystallography and two- or three-dimensionalcomputational modeling. Accordingly, a given antibody sequence may beplaced into a canonical class which allows for, among other things,identifying appropriate chassis sequences (e.g., based on a desire toinclude a variety of canonical structures in a library). Kabat numberingof antibody amino acid sequences and structural considerations asdescribed by Chothia et al., loc. cit. and their implications forconstruing canonical aspects of antibody structure, are described in theliterature. The subunit structures and three-dimensional configurationsof different classes of immunoglobulins are well known in the art. For areview of the antibody structure, see Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

The CDR3 of the light chain and, particularly, the CDR3 of the heavychain may constitute the most important determinants in antigen bindingwithin the light and heavy chain variable regions. In some antibodyconstructs, the heavy chain CDR3 appears to constitute the major area ofcontact between the antigen and the antibody. In vitro selection schemesin which CDR3 alone is varied can be used to vary the binding propertiesof an antibody or determine which residues contribute to the binding ofan antigen. Hence, CDR3 is typically the greatest source of moleculardiversity within the antibody-binding site. H3, for example, can be asshort as two amino acid residues or greater than 26 amino acids.

The sequence of antibody genes after assembly and somatic mutation ishighly varied, and these varied genes are estimated to encode 10¹⁰different antibody molecules (Immunoglobulin Genes, 2n^(d) ed., eds.Jonio et al., Academic Press, San Diego, Calif., 1995). Accordingly, theimmune system provides a repertoire of immunoglobulins. The term“repertoire” refers to at least one nucleotide sequence derived whollyor partially from at least one sequence encoding at least oneimmunoglobulin. The sequence(s) may be generated by rearrangement invivo of the V, D, and J segments of heavy chains, and the V and Jsegments of light chains. Alternatively, the sequence(s) can begenerated from a cell in response to which rearrangement occurs, e.g.,in vitro stimulation. Alternatively, part or all of the sequence(s) maybe obtained by DNA splicing, nucleotide synthesis, mutagenesis, andother methods, see, e.g., U.S. Pat. No. 5,565,332. A repertoire mayinclude only one sequence or may include a plurality of sequences,including ones in a genetically diverse collection.

The term “bispecific” as used herein refers to an antibody constructwhich is “at least bispecific”, i.e., it comprises at least a firstbinding domain and a second binding domain, wherein the first bindingdomain binds to one antigen or target, and the second binding domainbinds to another antigen or target (here: CD3). Accordingly, antibodyconstructs according to the invention comprise specificities for atleast two different antigens or targets. The term “bispecific antibodyconstruct” of the invention also encompasses multispecific antibodyconstructs such as trispecific antibody constructs, the latter onesincluding three binding domains, or constructs having more than three(e.g. four, five . . . ) specificities.

Given that the antibody constructs according to the invention are (atleast) bispecific, they do not occur naturally and they are markedlydifferent from naturally occurring products. A “bispecific” antibodyconstruct or immunoglobulin is hence an artificial hybrid antibody orimmunoglobulin having at least two distinct binding sites with differentspecificities. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990).

The at least two binding domains and the variable domains of theantibody construct of the present invention may or may not comprisepeptide linkers (spacer peptides). The term “peptide linker” defines inaccordance with the present invention an amino acid sequence by whichthe amino acid sequences of one (variable and/or binding) domain andanother (variable and/or binding) domain of the antibody construct ofthe invention are linked with each other. An essential technical featureof such peptide linker is that said peptide linker does not comprise anypolymerization activity. Among the suitable peptide linkers are thosedescribed in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344.

In the event that a linker is used, this linker is preferably of alength and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities. For peptide linkers which connectthe at least two binding domains in the antibody construct of theinvention (or two variable domains), those peptide linkers are preferredwhich comprise only a few number of amino acid residues, e.g. 12 aminoacid residues or less. Thus, peptide linker of 12, 11, 10, 9, 8, 7, 6 or5 amino acid residues are preferred. An envisaged peptide linker withless than 5 amino acids comprises 4, 3, 2 or one amino acid(s) whereinGly-rich linkers are preferred. A particularly preferred “single” aminoacid in context of said “peptide linker” is Gly. Accordingly, saidpeptide linker may consist of the single amino acid Gly. Anotherpreferred embodiment of a peptide linker is characterized by the aminoacid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly₄Ser, or polymers thereof,i.e. (Gly₄Ser)x, where x is an integer of 1 or greater. Thecharacteristics of said peptide linker, which comprise the absence ofthe promotion of secondary structures are known in the art and aredescribed e.g. in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273),Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow(FASEB (1995) 9(1), 73-80). Peptide linkers which also do not promoteany secondary structures are preferred. The linkage of said domains toeach other can be provided by, e.g. genetic engineering, as described inthe examples. Methods for preparing fused and operatively linkedbispecific single chain constructs and expressing them in mammaliancells or bacteria are well-known in the art (e.g. WO 99/54440 orSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York, 2001).

Bispecific single chain molecules are known in the art and are describedin WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS,(1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45,193-197, Löffler, Blood, (2000), 95, 6, 2098-2103, Brühl, Immunol.,(2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56.Techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778, Kontermann and Dübel (2010), loc.cit. and Little (2009), loc. cit.) can be adapted to produce singlechain antibody constructs specifically recognizing (an) electedtarget(s).

Bivalent (also called divalent) or bispecific single-chain variablefragments (bi-scFvs or di-scFvs having the format (scFv)₂) can beengineered by linking two scFv molecules. If these two scFv moleculeshave the same binding specificity, the resulting (scFv)₂ molecule willpreferably be called bivalent (i.e. it has two valences for the sametarget epitope). If the two scFv molecules have different bindingspecificities, the resulting (scFv)₂ molecule will preferably be calledbispecific. The linking can be done by producing a single peptide chainwith two VH regions and two VL regions, yielding tandem scFvs (see e.g.Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Anotherpossibility is the creation of scFv molecules with linker peptides thatare too short for the two variable regions to fold together (e.g. aboutfive amino acids), forcing the scFvs to dimerize. This type is known asdiabodies (see e.g. Hollinger, Philipp et al., (July 1993) Proceedingsof the National Academy of Sciences of the United States of America 90(14): 6444-8.).

Single domain antibodies comprise merely one (monomeric) antibodyvariable domain which is able to bind selectively to a specific antigen,independently of other V regions or domains. The first single domainantibodies were engineered from havy chain antibodies found in camelids,and these are called V_(H)H fragments. Cartilaginous fishes also haveheavy chain antibodies (IgNAR) from which single domain antibodiescalled V_(NAR) fragments can be obtained. An alternative approach is tosplit the dimeric variable domains from common immunoglobulins e.g. fromhumans or rodents into monomers, hence obtaining VH or VL as a singledomain Ab. Although most research into single domain antibodies iscurrently based on heavy chain variable domains, nanobodies derived fromlight chains have also been shown to bind specifically to targetepitopes. Examples of single domain antibodies are called sdAb,nanobodies or single variable domain antibodies.

A (single domain mAb)₂ is hence a monoclonal antibody construct composedof (at least) two single domain monoclonal antibodies, which areindividually selected from the group comprising VH, VL, V_(H)H andV_(NAR). The linker is preferably in the form of a peptide linker.Similarly, an “scFv-single domain mAb” is a monoclonal antibodyconstruct composed of at least one single domain antibody as describedabove and one scFv molecule as described above. Again, the linker ispreferably in the form of a peptide linker.

Bispecific single chain molecules are known in the art and are describedin WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS,(1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45,193-197, Löffler, Blood, (2000), 95, 6, 2098-2103, Brühl, Immunol.,(2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56.Techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778, Kontermann and Dübel (2010), loc.cit. and Little (2009), loc. cit.) can be adapted to produce singlechain antibody constructs specifically recognizing (an) electedtarget(s).

Bivalent (also called divalent) or bispecific single-chain variablefragments (bi-scFvs or di-scFvs having the format (scFv)₂) can beengineered by linking two scFv molecules. If these two scFv moleculeshave the same binding specificity, the resulting (scFv)₂ molecule willpreferably be called bivalent (i.e. it has two valences for the sametarget epitope). If the two scFv molecules have different bindingspecificities, the resulting (scFv)₂ molecule will preferably be calledbispecific. The linking can be done by producing a single peptide chainwith two VH regions and two VL regions, yielding tandem scFvs (see e.g.Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Anotherpossibility is the creation of scFv molecules with linker peptides thatare too short for the two variable regions to fold together (e.g. aboutfive amino acids), forcing the scFvs to dimerize. This type is known asdiabodies (see e.g. Hollinger, Philipp et al., (July 1993) Proceedingsof the National Academy of Sciences of the United States of America 90(14): 6444-8.).

Single domain antibodies comprise merely one (monomeric) antibodyvariable domain which is able to bind selectively to a specific antigen,independently of other V regions or domains. The first single domainantibodies were engineered from havy chain antibodies found in camelids,and these are called V_(H)H fragments. Cartilaginous fishes also haveheavy chain antibodies (IgNAR) from which single domain antibodiescalled V_(NAR) fragments can be obtained. An alternative approach is tosplit the dimeric variable domains from common immunoglobulins e.g. fromhumans or rodents into monomers, hence obtaining VH or VL as a singledomain Ab. Although most research into single domain antibodies iscurrently based on heavy chain variable domains, nanobodies derived fromlight chains have also been shown to bind specifically to targetepitopes. Examples of single domain antibodies are called sdAb,nanobodies or single variable domain antibodies.

A (single domain mAb)₂ is hence a monoclonal antibody construct composedof (at least) two single domain monoclonal antibodies, which areindividually selected from the group comprising VH, VL, V_(H)H andV_(NAR). The linker is preferably in the form of a peptide linker.Similarly, an “scFv-single domain mAb” is a monoclonal antibodyconstruct composed of at least one single domain antibody as describedabove and one scFv molecule as described above. Again, the linker ispreferably in the form of a peptide linker.

It is also envisaged that the antibody construct of the invention has,in addition to its function to bind to the target antigen and CD3, afurther function. In this format, the antibody construct is atrifunctional or multifunctional antibody construct by targeting targetcells through binding to the target antigen, mediating cytotoxic T cellactivity through CD3 binding and providing a further function such as alabel (fluorescent etc.), a therapeutic agent such as a toxin orradionuclide, etc.

Covalent modifications of the antibody constructs are also includedwithin the scope of this invention, and are generally, but not always,done post-translationally. For example, several types of covalentmodifications of the antibody construct are introduced into the moleculeby reacting specific amino acid residues of the antibody construct withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl andamino terminal residues are reacted with succinic or other carboxylicacid anhydrides. Derivatization with these agents has the effect ofreversing the charge of the lysinyl residues. Other suitable reagentsfor derivatizing alpha-amino-containing residues include imidoesterssuch as methyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking theantibody constructs of the present invention to a water-insolublesupport matrix or surface for use in a variety of methods. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the antibody constructsincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody construct isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the amino acid sequence of an antibody construct is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody construct is by chemical or enzymatic coupling of glycosides tothe protein. These procedures are advantageous in that they do notrequire production of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330, and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

Removal of carbohydrate moieties present on the starting antibodyconstruct may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Other modifications of the antibody construct are contemplated herein.For example, another type of covalent modification of the antibodyconstruct comprises linking the antibody construct to variousnon-proteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, as is knownin the art, amino acid substitutions may be made in various positionswithin the antibody construct, e.g. in order to facilitate the additionof polymers such as PEG.

In some embodiments, the covalent modification of the antibodyconstructs of the invention comprises the addition of one or morelabels. The labelling group may be coupled to the antibody construct viaspacer arms of various lengths to reduce potential steric hindrance.Various methods for labelling proteins are known in the art and can beused in performing the present invention. The term “label” or “labellinggroup” refers to any detectable label. In general, labels fall into avariety of classes, depending on the assay in which they are to bedetected—the following examples include, but are not limited to:

-   -   a) isotopic labels, which may be radioactive or heavy isotopes,        such as radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S,        ⁸⁹Z, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I)    -   b) magnetic labels (e.g., magnetic particles)    -   c) redox active moieties    -   d) optical dye (including, but not limited to, chromophores,        phosphors and fluorophores) such as fluorescent groups (e.g.,        FITC, rhodamine, lanthanide phosphors), chemiluminescent groups,        and fluorophores which can be either “small molecule” fluores or        proteinaceous fluores    -   e) enzymatic groups (e.g. horseradish peroxidase,        β-galactosidase, luciferase, alkaline phosphatase)    -   f) biotinylated groups    -   g) predetermined polypeptide epitopes recognized by a secondary        reporter (e.g., leucine zipper pair sequences, binding sites for        secondary antibodies, metal binding domains, epitope tags, etc.)

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR),FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7(Amersham Life Science, Pittsburgh, Pa.). Suitable optical dyes,including fluorophores, are described in Molecular Probes Handbook byRichard P. Haugland.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558).

Leucine zipper domains are peptides that promote oligomerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., 1988,Science 240:1759), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble oligomericproteins are described in PCT application WO 94/10308, and the leucinezipper derived from lung surfactant protein D (SPD) described in Hoppeet al., 1994, FEBS Letters 344:191. The use of a modified leucine zipperthat allows for stable trimerization of a heterologous protein fusedthereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78.In one approach, recombinant fusion proteins comprising the targetantigen antibody fragment or derivative fused to a leucine zipperpeptide are expressed in suitable host cells, and the soluble oligomerictarget antigen antibody fragments or derivatives that form are recoveredfrom the culture supernatant.

The antibody construct of the invention may also comprise additionaldomains, which are e.g. helpful in the isolation of the molecule orrelate to an adapted pharmacokinetic profile of the molecule. Domainshelpful for the isolation of an antibody construct may be selected frompeptide motives or secondarily introduced moieties, which can becaptured in an isolation method, e.g. an isolation column. Non-limitingembodiments of such additional domains comprise peptide motives known asMyc-tag, HAT-tag, HA-tag, TAP-tag, GST-tag, chitin binding domain(CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag andvariants thereof (e.g. StrepII-tag) and His-tag. All herein disclosedantibody constructs characterized by the identified CDRs are preferredto comprise a His-tag domain, which is generally known as a repeat ofconsecutive His residues in the amino acid sequence of a molecule,preferably of six His residues.

T cells or T lymphocytes are a type of lymphocyte (itself a type ofwhite blood cell) that play a central role in cell-mediated immunity.There are several subsets of T cells, each with a distinct function. Tcells can be distinguished from other lymphocytes, such as B cells andNK cells, by the presence of a T cell receptor (TCR) on the cellsurface. The TCR is responsible for recognizing antigens bound to majorhistocompatibility complex (MHC) molecules and is composed of twodifferent protein chains. In 95% of the T cells, the TCR consists of analpha (α) and beta (β) chain. When the TCR engages with antigenicpeptide and MHC (peptide/MHC complex), the T lymphocyte is activatedthrough a series of biochemical events mediated by associated enzymes,co-receptors, specialized adaptor molecules, and activated or releasedtranscription factors

The CD3 receptor complex is a protein complex and is composed of fourchains. In mammals, the complex contains a CD3γ (gamma) chain, a CD3δ(delta) chain, and two CD3ε (epsilon) chains. These chains associatewith the T cell receptor (TCR) and the so-called ζ (zeta) chain to formthe T cell receptor CD3 complex and to generate an activation signal inT lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chainsare highly related cell-surface proteins of the immunoglobulinsuperfamily containing a single extracellular immunoglobulin domain. Theintracellular tails of the CD3 molecules contain a single conservedmotif known as an immunoreceptor tyrosine-based activation motif or ITAMfor short, which is essential for the signaling capacity of the TCR. TheCD3 epsilon molecule is a polypeptide which in humans is encoded by theCD3ε gene which resides on chromosome 11. The sequence of a preferredhuman CD3 epsilon extracellular domain is shown in SEQ ID NO: 605, andthe most preferred CD3 binding epitope corresponding to amino acidresidues 1-27 of the human CD3 epsilon extracellular domain isrepresented in SEQ ID NO: 604.

The redirected lysis of target cells via the recruitment of T cells by amultispecific, at least bispecific, antibody construct involvescytolytic synapse formation and delivery of perforin and granzymes. Theengaged T cells are capable of serial target cell lysis, and are notaffected by immune escape mechanisms interfering with peptide antigenprocessing and presentation, or clonal T cell differentiation; see, forexample, WO 2007/042261.

Cytotoxicity mediated by bispecific antibody constructs can be measuredin various ways. Effector cells can be e.g. stimulated enriched (human)CD8 positive T cells or unstimulated (human) peripheral bloodmononuclear cells (PBMC). If the target cells are of macaque origin orexpress or are transfected with macaque target cell antigen, theeffector cells should also be of macaque origin such as a macaque T cellline, e.g. 4119LnPx. The target cells should express (at least theextracellular domain of) target cell antigen, e.g. human or macaquetarget cell antigen. Target cells can be a cell line (such as CHO) whichis stably or transiently transfected with target cell antigen, e.g.human or macaque target cell antigen. Alternatively, the target cellscan be a target cell antigen positive natural expresser cell line, suchas a human cancer cell line. Usually EC50 values are expected to belower with target cell lines expressing higher levels of target cellantigen on the cell surface. The effector to target cell (E:T) ratio isusually about 10:1, but can also vary. Cytotoxic activity of bispecificantibody constructs can be measured in a ⁵¹chromium release assay(incubation time of about 18 hours) or in a in a FACS-based cytotoxicityassay (incubation time of about 48 hours). Modifications of the assayincubation time (cytotoxic reaction) are also possible. Other methods ofmeasuring cytotoxicity are well-known to the skilled person and compriseMTT or MTS assays, ATP-based assays including bioluminescent assays, thesulforhodamine B (SRB) assay, WST assay, clonogenic assay and the ECIStechnology.

The cytotoxic activity mediated by bispecific antibody constructs of thepresent invention is preferably measured in a cell-based cytotoxicityassay. It is represented by the EC₅₀ value, which corresponds to thehalf maximal effective concentration (concentration of the antibodyconstruct which induces a cytotoxic response halfway between thebaseline and maximum). Preferably, the EC₅₀ value of the bispecificantibody constructs is ≦20,000 pg/ml, more preferably ≦5000 pg/ml, evenmore preferably ≦1000 pg/ml, even more preferably 5500 pg/ml, even morepreferably ≦350 pg/ml, even more preferably ≦250 pg/ml, even morepreferably ≦100 pg/ml, even more preferably ≦50 pg/ml, even morepreferably ≦10 pg/ml, and most preferably ≦5 pg/ml.

Any of the above given EC₅₀ values can be combined with any one of theindicated scenarios of a cell-based cytotoxicity assay, e.g. in linewith the methods described in the appended example. For example, when(human) CD8 positive T cells or a macaque T cell line are used aseffector cells, the EC₅₀ value of the bispecific antibody construct ofthe invention (e.g. a target cell antigen/CD3 bispecific construct) ispreferably ≦1000 pg/ml, more preferably ≦500 pg/ml, even more preferably≦250 pg/ml, even more preferably ≦100 pg/ml, even more preferably ≦50pg/ml, even more preferably ≦10 pg/ml, and most preferably ≦5 pg/ml. Ifin this assay the target cells are (human or macaque) cells transfectedwith the target antigen (e.g. target cell antigen), such as CHO cells,the EC₅₀ value of the bispecific antibody construct is preferably ≦150pg/ml, more preferably ≦100 pg/ml, even more preferably 550 pg/ml, evenmore preferably ≦30 pg/ml, even more preferably ≦10 pg/ml, and mostpreferably ≦5 pg/ml. If the target cells are a positive naturalexpresser cell line (e.g. of target cell antigen), then the EC₅₀ valueis preferably ≦350 pg/ml, more preferably ≦250 pg/ml, even morepreferably ≦200 pg/ml, even more preferably ≦100 pg/ml, even morepreferably ≦150 pg/ml, even more preferably ≦100 pg/ml, and mostpreferably ≦50 pg/ml, or lower. When (human) PBMCs are used as effectorcells, the EC₅₀ value of the bispecific antibody construct is preferably≦1000 pg/ml, more preferably ≦750 pg/ml, more preferably ≦500 pg/ml,even more preferably ≦350 pg/ml, even more preferably ≦250 pg/ml, evenmore preferably ≦100 pg/ml, and most preferably ≦50 pg/ml, or lower.

Preferably, the bispecific antibody constructs of the present inventiondo not induce/mediate lysis or do not essentially induce/mediate lysisof target cell antigen negative cells such as CHO cells. The term “donot induce lysis”, “do not essentially induce lysis”, “do not mediatelysis” or “do not essentially mediate lysis” means that an antibodyconstructs of the present invention does not induce or mediate lysis ofmore than 30%, preferably not more than 20%, more preferably not morethan 10%, particularly preferably not more than 9%, 8%, 7%, 6% or 5% oftarget cell antigen negative cells, whereby lysis of a target cellantigen positive cell line is set to be 100%. This usually applies forconcentrations of the antibody construct of up to 500 nM. The skilledperson knows how to measure cell lysis without further ado. Moreover,the present specification teaches specific instructions how to measurecell lysis.

The difference in cytotoxic activity between the monomeric and thedimeric isoform of individual bispecific antibody constructs is referredto as “potency gap”. This potency gap can e.g. be calculated as ratiobetween EC₅₀ values of the molecule's monomeric and dimeric form.Potency gaps of the bispecific antibody constructs of the presentinvention are preferably ≦5, more preferably ≦4, even more preferably≦3, even more preferably ≦2 and most preferably ≦1.

As well as the second binding domain the first (or any further) bindingdomain(s) of the antibody construct of the invention is/are preferablycross-species specific for members of the mammalian order of primates.Cross-species specific CD3 binding domains are, for example, describedin WO 2008/119567. According to one embodiment, the first and secondbinding domain, in addition to binding to human target cell antigen andhuman CD3, respectively, will also bind to the target cell antigen/CD3of primates including (but not limited to) new world primates (such asCallithrix jacchus, Saguinus Oedipus or Saimiri sciureus), old worldprimates (such baboons and macaques), gibbons, and non-human homininae.

In one aspect of the invention, the first binding domain binds to humanCDH19 (SEQ ID NO: 606) and further binds to macaque CDH19, such as CDH19of Macaca fascicularis (SEQ ID NO: 607). The affinity of the firstbinding domain for macaque CDH19 is preferably 515 nM, more preferably≦10 nM, even more preferably ≦5 nM, even more preferably ≦1 nM, evenmore preferably ≦0.5 nM, even more preferably ≦0.1 nM, and mostpreferably ≦0.05 nM or even ≦0.01 nM.

Preferably the affinity gap of the antibody constructs according to theinvention for binding to its macaque vs human target antigen, e.g.macaque CDH19 versus human CDH19 [maCDH19:huCDH19] is between 0.1 and10, more preferably between 0.2 and 5, even more preferably between 0.3and 2.5, even more preferably between 0.4 and 2, and most preferablybetween 0.5 and 1.

In one embodiment the invention provides a bispecific single chainantibody construct, wherein serum albumin is a human serum albumin or anFcRn binding optimized variant thereof. Variants of human albumin aredescribed e.g. in WO 2014/072481.

In one embodiment of the antibody construct of the invention, the serumalbumin comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4 to 12 and 608 to 628.

Also in one embodiment of the antibody construct of the invention, theserum albumin is linked to the antibody construct via a peptide linker.It is preferred that the peptide linker has the amino acid sequence(GGGGS)_(n) (SEQ ID NO: 13)_(n), (PGGGGS)_(n) (SEQ ID NO: 2707)_(n), or(PGGDGS)_(n) (SEQ ID NO: 2708)_(n), wherein “n” is an integer in therange of 1 to 5. Further preferred is that “n” is an integer in therange of 1 to 3, and most preferably “n” is 1 or 2.

In one embodiment of the antibody construct of the invention the secondbinding domain comprises a VL region having CDR-L1-L3 and a VH regionhaving CDR-H1 -H3 selected from the group consisting of:

-   -   (a) CDR-L1-L3 as depicted in SEQ ID NOs:14-16 and CDR-H1-H3 as        depicted in SEQ ID NOs:17-19;    -   (b) CDR-L1-L3 as depicted in SEQ ID NOs:26-28 and CDR-H1-H3 as        depicted in SEQ ID NOs:29-31;    -   (c) CDR-L1-L3 as depicted in SEQ ID NOs:38-40 and CDR-H1-H3 as        depicted in SEQ ID NOs:41-43;    -   (d) CDR-L1-L3 as depicted in SEQ ID NOs:50-52 and CDR-H1-H3 as        depicted in SEQ ID NOs:53-55;    -   (e) CDR-L1-L3 as depicted in SEQ ID NOs:62-64 and CDR-H1-H3 as        depicted in SEQ ID NOs:65-67;    -   (f) CDR-L1-L3 as depicted in SEQ ID NOs:74-76 and CDR-H1-H3 as        depicted in SEQ ID NOs:77-79;    -   (g) CDR-L1-L3 as depicted in SEQ ID NOs:86-88 and CDR-H1-H3 as        depicted in SEQ ID NOs:89-91;    -   (h) CDR-L1-L3 as depicted in SEQ ID NOs:98-100 and CDR-H1-H3 as        depicted in SEQ ID NOs:101-103;    -   (i) CDR-L1-L3 as depicted in SEQ ID NOs:110-112 and CDR-H1-H3 as        depicted in SEQ ID NOs:113-115; and    -   (j) CDR-L1-L3 as depicted in SEQ ID NOs:122-124 and CDR-H1-H3 as        depicted in SEQ ID NOs:125-127.

In one embodiment of the antibody construct of the invention the secondbinding domain comprises pairs of VH and VL chains selected from thegroup consisting of:

-   -   (a) a VH-chain as depicted in SEQ ID NO: 20 and a VL-chain as        depicted in SEQ ID NO: 22;    -   (b) a VH-chain as depicted in SEQ ID NO: 32 and a VL-chain as        depicted in SEQ ID NO: 34;    -   (c) a VH-chain as depicted in SEQ ID NO: 44 and a VL-chain as        depicted in SEQ ID NO: 46;    -   (d) a VH-chain as depicted in SEQ ID NO: 56 and a VL-chain as        depicted in SEQ ID NO: 58;    -   (e) a VH-chain as depicted in SEQ ID NO: 68 and a VL-chain as        depicted in SEQ ID NO: 70;    -   (f) a VH-chain as depicted in SEQ ID NO: 80 and a VL-chain as        depicted in SEQ ID NO: 82;    -   (g) a VH-chain as depicted in SEQ ID NO: 92 and a VL-chain as        depicted in SEQ ID NO: 94;    -   (h) a VH-chain as depicted in SEQ ID NO: 104 and a VL-chain as        depicted in SEQ ID NO: 106;    -   (i) a VH-chain as depicted in SEQ ID NO: 116 and a VL-chain as        depicted in SEQ ID NO: 118; and    -   (j) a VH-chain as depicted in SEQ ID NO: 128 and a VL-chain as        depicted in SEQ ID NO: 130.

Also in one embodiment of the antibody construct of the invention thesecond binding domain comprises an amino acid sequence as depicted inSEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 48, SEQ ID NO: 60, SEQ ID NO:72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120 or SEQID NO: 132.

In a further embodiment of the antibody construct of the invention, thefirst binding domain binds to a target cell surface antigen, which is atumor antigen or a viral antigen on the surface of an infected hostcell. In a preferred embodiment the tumor antigen is selected from thegroup consisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, CD20,and CD70. Preferred viral antigens are selected from a group consistingof Influenza A, CMV and HIV.

The invention further provides an antibody construct, wherein theantibody construct comprises an amino acid sequences selected from thegroup consisting of:

-   -   (a) SEQ ID NOs: 134-180;    -   (b) SEQ ID NOs: 181-227;    -   (c) SEQ ID NOs: 228-274;    -   (d) SEQ ID NOs: 275-321;    -   (e) SEQ ID NOs: 322-368;    -   (f) SEQ ID NOs: 369-415;    -   (g) SEQ ID NOs: 416-462;    -   (h) SEQ ID NOs: 463-509;    -   (i) SEQ ID NOs: 510-556; and    -   (j) SEQ ID NOs: 557-603.

The invention also provides an antibody construct, wherein the antibodyconstruct comprises the following elements starting from the N-terminus:

-   -   (a) an scFv binding to the target cell surface antigen having an        amino acid sequence selected from the group consisting of SEQ ID        NOs: 629-675, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774,        784, 794, 804, 814, 824, 834, 844, 854, 864, 874, 884, 894, 904,        914, 924, 934, 944, 954, 964, 974, 984, 994, 1004, 1014, 1024,        1034, 1044, 1054, 1064, 1074, 1084, 1094, 1104, 1114, 1124,        1134, 1144, 1154, 1164, 1174, 1184, 1194, 1204, 1214, 1224,        1234, 1244, 1254, 1264, 1274, 1284, 1294, 1304, 1314, 1324,        1334, 1344, 1354, 1364, 1374, 1384, 1394, 1404, 1414, 1424,        1434, 1444, 1454, 1464, 1474, 1484, 1494, 1504, 1514, 1524,        1534, 1544, 1554, 1564, 1574, 1584, 1594, 1604, 1614, 1624,        1634, 1644, 1654, 1664, 1674, 1684, 1694, 1704, 1714, 1724,        1734, 1744, 1754, 1764, 1774, 1784, 1794, 1804, 1814, 1824,        1834, 1844, 1854, 1864, 1874, 1884, 1894, 1904, 1914, 1924,        1934, 1944, 1954, 1964, 1974, 2004, 2014, 2024, 2034, 2044,        2054, 2064, 2074, 2084, 2094, 2104, 2114, 2124, 2134, 2144,        2154, 2164, 2174, 2184, 2194, 2204, 2214, 2224, 2234, 2244,        2254, 2264, 2274, 2284, 2294, 2304, 2314, 2324, 2334, 2344,        2354, 2364, 2374, 2384, 2394, 2404, 2414, 2424, 2434, 2444,        2454, 2464, 2474, 2484, 2494, 2504, 2514, 2524, 2534, 2544,        2554, 2564, 2574, 2584, 2594, 2604, 2614, 2624, 2634, 2644,        2654, 2664, 2674, 2684, 2694, and 2704;    -   (b) a peptide linker having an amino acid sequence selected from        the group consisting of

SEQ ID NOs: 13 and 2707-2709;

-   -   (c) an scFv binding to the T cell surface antigen CD3 having an        amino acid sequence selected from the group consisting of SEQ ID        NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132 and 2706;    -   (d) a peptide linker having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 13 and 2707-2709;    -   (e) a serum albumin having an amino acid sequence selected from        the group consisting of

SEQ ID NOs: 4 to 12 and 608 to 628; and

-   -   (f) optionally a His-tag.

It is particularly preferred that the elements defined in above items(a) to (f) are arranged in this sequence from the N-terminus to theC-terminus of the antibody construct.

Amino acid sequence modifications of the antibody constructs describedherein are also contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody construct. Amino acid sequence variants of the antibodyconstructs are prepared by introducing appropriate nucleotide changesinto the antibody constructs nucleic acid, or by peptide synthesis. Allof the below described amino acid sequence modifications should resultin an antibody construct which still retains the desired biologicalactivity (binding to target cell antigen and to CD3) of the unmodifiedparental molecule.

The term “amino acid” or “amino acid residue” typically refers to anamino acid having its art recognized definition such as an amino acidselected from the group consisting of: alanine (Ala or A); arginine (Argor R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys orC); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G);histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine(Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line(Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp orW); tyrosine (Tyr or Y); and valine (Val or V), although modified,synthetic, or rare amino acids may be used as desired. Generally, aminoacids can be grouped as having a nonpolar side chain (e.g., Ala, Cys,He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g.,Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or anuncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe,Ser, Thr, Trp, and Tyr).

Amino acid modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the antibody constructs. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody constructs, such as changing the number or position ofglycosylation sites.

For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or deletedin each of the CDRs (of course, dependent on their length), while 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25amino acids may be inserted or deleted in each of the FRs. Preferably,amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residuesto polypeptides containing a hundred or more residues, as well asintra-sequence insertions of single or multiple amino acid residues. Aninsertional variant of the antibody construct of the invention includesthe fusion to the N-terminus or to the C-terminus of the antibodyconstruct to an enzyme or a fusion to a polypeptide which increases theserum half-life of the antibody construct.

The sites of greatest interest for substitutional mutagenesis includethe CDRs of the heavy and/or light chain, in particular thehypervariable regions, but FR alterations in the heavy and/or lightchain are also contemplated. The substitutions are preferablyconservative substitutions as described herein. Preferably, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids may be substituted in a CDR, while 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or25 amino acids may be substituted in the framework regions (FRs),depending on the length of the CDR or FR. For example, if a CDR sequenceencompasses 6 amino acids, it is envisaged that one, two or three ofthese amino acids are substituted. Similarly, if a CDR sequenceencompasses 15 amino acids it is envisaged that one, two, three, four,five or six of these amino acids are substituted.

A useful method for identification of certain residues or regions of theantibody constructs that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244: 1081-1085 (1989). Here, a residue or group oftarget residues within the antibody construct is/are identified (e.g.charged residues such as arg, asp, his, lys, and glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with theepitope.

Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site or regionfor introducing an amino acid sequence variation is predetermined, thenature of the mutation per se needs not to be predetermined. Forexample, to analyze or optimize the performance of a mutation at a givensite, alanine scanning or random mutagenesis may be conducted at atarget codon or region, and the expressed antibody construct variantsare screened for the optimal combination of desired activity. Techniquesfor making substitution mutations at predetermined sites in the DNAhaving a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of target antigen binding activities.

Generally, if amino acids are substituted in one or more or all of theCDRs of the heavy and/or light chain, it is preferred that thethen-obtained “substituted” sequence is at least 60%, more preferably65%, even more preferably 70%, particularly preferably 75%, moreparticularly preferably 80% identical to the “original” CDR sequence.This means that it is dependent of the length of the CDR to which degreeit is identical to the “substituted” sequence. For example, a CDR having≦amino acids is preferably 80% identical to its substituted sequence inorder to have at least one amino acid substituted. Accordingly, the CDRsof the antibody construct may have different degrees of identity totheir substituted sequences, e.g., CDRL1 may have 80%, while CDRL3 mayhave 90%.

Preferred substitutions (or replacements) are conservativesubstitutions. However, any substitution (including non-conservativesubstitution or one or more from the “exemplary substitutions” listed inTable 1, below) is envisaged as long as the antibody construct retainsits capability to bind to target cell antigen via the first bindingdomain and to CD3 epsilon via the second binding domain and/or its CDRshave an identity to the then substituted sequence (at least 60%, morepreferably 65%, even more preferably 70%, particularly preferably 75%,more particularly preferably 80% identical to the “original” CDRsequence).

Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table A, or as further described below inreference to amino acid classes, may be introduced and the productsscreened for a desired characteristic.

TABLE A Amino acid substitutions Preferred Original ExemplarySubstitutions Substitutions Ala (A) val, leu, ile val Arg (R) lys, gln,asn lys Asn (N) gln, his, asp, lys, arg gln Asp (D) glu, asn glu Cys (C)ser, ala ser Gln (Q) asn, glu asn Glu (E) asp, gln asp Gly (G) Ala alaHis (H) asn, gln, lys, arg arg Ile (I) leu, val, met, ala, phe leu Leu(L) norleucine, ile, val, met, ala ile Lys (K) arg, gln, asn arg Met (M)leu, phe, ile leu Phe (F) leu, val, ile, ala, tyr tyr Pro (P) Ala alaSer (S) Thr thr Thr (T) Ser ser Trp (W) tyr, phe tyr Tyr (Y) trp, phe,thr, ser phe Val (V) ile, leu, met, phe, ala leu

Substantial modifications in the biological properties of the antibodyconstruct of the present invention are accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues are divided intogroups based on common side-chain properties: (1) hydrophobic:norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser,thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5)residues that influence chain orientation: gly, pro; and (6) aromatic:trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the antibody construct may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant crosslinking. Conversely, cysteinebond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Eva 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;Xu set to 16, and Xg set to 40 for database search stage and to 67 forthe output stage of the algorithms. Gapped alignments are triggered by ascore corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs are at least 60% to the sequences depictedherein, and more typically with preferably increasing homologies oridentities of at least 65% or 70%, more preferably at least 75% or 80%,even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, and almost 100%. In a similar manner, “percent (%)nucleic acid sequence identity” with respect to the nucleic acidsequence of the binding proteins identified herein is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues in the coding sequence of theantibody construct. A specific method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andthe nucleotide sequences depicted herein are at least 60%, and moretypically with preferably increasing homologies or identities of atleast 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.Thus, a “variant CDR” is one with the specified homology, similarity, oridentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity ofthe parent CDR.

It is furthermore envisaged that the bispecific antibody constructs ofthe present invention exhibit therapeutic efficacy or anti-tumoractivity. This can e.g. be assessed in a study as disclosed in thefollowing example of an advanced stage human tumor xenograft model:

On day 1 of the study, 5×10⁶ cells of a human target cell antigenpositive cancer cell line are subcutaneously injected in the rightdorsal flank of female NOD/SCID mice. When the mean tumor volume reachesabout 100 mm³, in vitro expanded human CD3 positive T cells aretransplanted into the mice by injection of about 2×10⁷ cells into theperitoneal cavity of the animals. Mice of vehicle control group 1 do notreceive effector cells and are used as an untransplanted control forcomparison with vehicle control group 2 (receiving effector cells) tomonitor the impact of T cells alone on tumor growth. The antibodytreatment starts when the mean tumor volume reaches about 200 mm³. Themean tumor size of each treatment group on the day of treatment startshould not be statistically different from any other group (analysis ofvariance). Mice are treated with 0.5 mg/kg/day of a target cellantigen/CD3 bispecifc antibody construct by intravenous bolus injectionfor about 15 to 20 days. Tumors are measured by caliper during the studyand progress evaluated by intergroup comparison of tumor volumes (TV).The tumor growth inhibition T/C [%] is determined by calculating TV asT/C% =100×(median TV of analyzed group)/(median TV of control group 2).

The skilled person knows how to modify or adapt certain parameters ofthis study, such as the number of injected tumor cells, the site ofinjection, the number of transplanted human T cells, the amount ofbispecific antibody constructs to be administered, and the timelines,while still arriving at a meaningful and reproducible result.Preferably, the tumor growth inhibition T/C [%] is ≦70 or ≦60, morepreferably ≦50 or ≦40, even more preferably ≦30 or ≦20 and mostpreferably ≦10 or ≦5 or even ≦2.5.

In one embodiment, the bispecific antibody constructs of the presentinvention exhibit high monomer yields under standard research scaleconditions, e.g., in a standard two-step purification process.Preferably the monomer yield of the antibody constructs according to theinvention is ≧0.25 mg/L supernatant, more preferably ≧0.5 mg/L, evenmore preferably ≧1 mg/L, and most preferably ≧3 mg/L supernatant.

Likewise, the yield of the dimeric antibody construct isoforms and themonomer percentage (Le., monomer : (monomer+dimer)) of the antibodyconstructs can be determined. The productivity of monomeric and dimericantibody constructs and the calculated monomer percentage can e.g. beobtained in the SEC purification step of culture supernatant fromstandardized research-scale production in roller bottles. In oneembodiment, the monomer percentage of the antibody constructs is ≧80%,more preferably ≧85%, even more preferably ≧90%, and most preferably≧95%.

In a further embodiment, the percentage of identity to human germline ofthe antibody constructs according to the invention is ≧70% or ≧75%, morepreferably ≧80% or ≧85%, even more preferably ≧90%, and most preferably≧95%. Identity to human antibody germline gene products is thought to bean important feature to reduce the risk of therapeutic proteins toelicit an immune response against the drug in the patient duringtreatment. Hwang & Foote (“Immunogenicity of engineered antibodies”;Methods 36 (2005) 3-10) demonstrate that the reduction of non-humanportions of drug antibody constructs leads to a decrease of risk toinduce anti-drug antibodies in the patients during treatment. Bycomparing an exhaustive number of clinically evaluated antibody drugsand the respective immunogenicity data, the trend is shown thathumanization of the V-regions of antibodies makes the protein lessimmunogenic (average 5.1% of patients) than antibodies carryingunaltered non-human V regions (average 23.59% of patients). A higherdegree of identity to human sequences is hence desirable for V-regionbased protein therapeutics in the form of antibody constructs. For thispurpose of determining the germline identity, the V-regions of VL can bealigned with the amino acid sequences of human germline V segments and Jsegments (http://vbase.mrc-cpe.cam.ac.uk) using Vector NTI software andthe amino acid sequence calculated by dividing the identical amino acidresidues by the total number of amino acid residues of the VL inpercent. The same can be for the VH segments(http://vbase.mrc-cpe.cam.ac.uk/) with the exception that the VH CDR3may be excluded due to its high diversity and a lack of existing humangermline VH CDR3 alignment partners.

Recombinant techniques can then be used to increase sequence identity tohuman antibody germline genes.

In one embodiment, the antibody constructs have a preferred plasmastability (ratio of EC50 with plasma to EC50 w/o plasma) of ≦5, morepreferably ≦4, even more preferably ≦3, and most preferably ≦2. Theplasma stability of an antibody construct can be tested by incubation ofthe construct in human plasma at 37° C. for 24 hours followed by EC50determination in a 51chromium release cytotoxicity assay. The effectorcells in the cytotoxicity assay can be stimulated enriched human CD8positive T cells. Target cells can e.g. be CHO cells transfected withthe target antigen, e.g., CDH19. The effector to target cell (E:T) ratiocan be chosen as 10:1. The human plasma pool used for this purpose isderived from the blood of healthy donors collected by EDTA coatedsyringes. Cellular components are removed by centrifugation and theupper plasma phase is collected and subsequently pooled. As control,antibody constructs are diluted immediately prior to the cytotoxicityassay in RPMI-1640 medium. The plasma stability is calculated as ratioof EC50 (after plasma incubation) to EC50 (control).

It is preferred that the monomer to dimer conversion of antibodyconstructs of the invention is low. The conversion can be measured underdifferent conditions and analyzed by high performance size exclusionchromatography. For example, incubation of the monomeric isoforms of theantibody constructs can be carried out for 7 days at 37° C. andconcentrations of e.g. 100 μg/ml or 250 μg/ml in an incubator. Underthese conditions, it is preferred that the antibody constructs of theinvention show a dimer percentage that is ≦5%, more preferably ≦4%, evenmore preferably ≦3%, even more preferably ≦2.5%, even more preferably≦2%, even more preferably ≦1.5%, and most preferably ≦1%.

It is also preferred that the bispecific antibody constructs of thepresent invention present with very low dimer conversion after a numberof freeze/thaw cycles. For example, the antibody construct monomer isadjusted to a concentration of 250 μg/ml e.g. in SEC running buffer andsubjected to three freeze/thaw cycles (freezing at −80° C. for 30 minfollowed by thawing for 30 min at room temperature), followed by highperformance SEC to determine the percentage of initially monomericantibody construct, which had been converted into dimeric antibodyconstruct. Preferably the dimer percentages of the bispecific antibodyconstructs are ≦5%, more preferably ≦4%, even more preferably ≦3%, evenmore preferably ≦2.5%, even more preferably ≦2%, even more preferably≦1.5%, and most preferably ≦1%, for example after three freeze/thawcycles.

The bispecific antibody constructs of the present invention preferablyshow a favorable thermostability with melting temperatures above 60° C.This parameter can be determined as follows: Temperature melting curvesare determined by Differential Scanning calorimetry (DSC) to determineintrinsic biophysical protein stabilities of the antibody constructs.These experiments are performed using a MicroCal LLC (Northampton, MA,U.S.A) VP-DSC device. The energy uptake of a sample containing anantibody construct is recorded from 20° C. to 90° C. compared to asample containing only the formulation buffer. The antibody constructsare adjusted to a final concentration of 250 μg/ml e.g. in SEC runningbuffer. For recording of the respective melting curve, the overallsample temperature is increased stepwise. At each temperature T energyuptake of the sample and the formulation buffer reference is recorded.The difference in energy uptake Cp (kcal/mole/° C.) of the sample minusthe reference is plotted against the respective temperature. The meltingtemperature is defined as the temperature at the first maximum of energyuptake.

In a further embodiment the antibody construct according to theinvention is stable at acidic pH. The more tolerant the antibodyconstruct behaves at unphysiologic pH such as pH 5.5 (a pH which isrequired to run e.g. a cation exchange chromatography), the higher isthe recovery of the antibody construct eluted from an ion exchangecolumn relative to the total amount of loaded protein. Recovery of theantibody construct from an ion (e.g., cation) exchange column at pH 5.5is preferably ≧30%, more preferably ≧40%, more preferably ≧50%, evenmore preferably ≧60%, even more preferably ≧70%, even more preferably≧80%, and most preferably ≧90%.

It is furthermore envisaged that the bispecific antibody constructs ofthe present invention exhibit therapeutic efficacy or anti-tumoractivity. This can e.g. be assessed in a study as disclosed in thefollowing example of an advanced stage human tumor xenograft model.

In an alternative embodiment the invention provides a polynucleotideencoding an antibody construct of the invention.

A polynucleotide is a biopolymer composed of 13 or more nucleotidemonomers covalently bonded in a chain. DNA (such as cDNA) and RNA (suchas mRNA) are examples of polynucleotides with distinct biologicalfunction. Nucleotides are organic molecules that serve as the monomersor subunits of nucleic acid molecules like DNA or RNA. The nucleic acidmolecule or polynucleotide can be double stranded and single stranded,linear and circular. It is preferably comprised in a vector which ispreferably comprised in a host cell. Said host cell is, e.g. aftertransformation or transfection with the vector or the polynucleotide ofthe invention, capable of expressing the antibody construct. For thatpurpose the polynucleotide or nucleic acid molecule is operativelylinked with control sequences.

The genetic code is the set of rules by which information encoded withingenetic material (nucleic acids) is translated into proteins. Biologicaldecoding in living cells is accomplished by the ribosome which linksamino acids in an order specified by mRNA, using tRNA molecules to carryamino acids and to read the mRNA three nucleotides at a time. The codedefines how sequences of these nucleotide triplets, called codons,specify which amino acid will be added next during protein synthesis.With some exceptions, a three-nucleotide codon in a nucleic acidsequence specifies a single amino acid. Because the vast majority ofgenes are encoded with exactly the same code, this particular code isoften referred to as the canonical or standard genetic code. While thegenetic code determines the protein sequence for a given coding region,other genomic regions can influence when and where these proteins areproduced.

In a further alternative embodiment the invention provides a vectorcomprising a polynucleotide of the invention.

A vector is a nucleic acid molecule used as a vehicle to transfer(foreign) genetic material into a cell. The term “vector”encompasses—but is not restricted to—plasmids, viruses, cosmids andartificial chromosomes. In general, engineered vectors comprise anorigin of replication, a multicloning site and a selectable marker. Thevector itself is generally a nucleotide sequence, commonly a DNAsequence, that comprises an insert (transgene) and a larger sequencethat serves as the “backbone” of the vector. Modern vectors mayencompass additional features besides the transgene insert and abackbone: promoter, genetic marker, antibiotic resistance, reportergene, targeting sequence, protein purification tag. Vectors calledexpression vectors (expression constructs) specifically are for theexpression of the transgene in the target cell, and generally havecontrol sequences.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Transfection” is the process of deliberately introducing nucleic acidmolecules or polynucleotides (including vectors) into target cells. Theterm is mostly used for non-viral methods in eukaryotic cells.Transduction is often used to describe virus-mediated transfer ofnucleic acid molecules or polynucleotides. Transfection of animal cellstypically involves opening transient pores or “holes” in the cellmembrane, to allow the uptake of material. Transfection can be carriedout using calcium phosphate, by electroporation, by cell squeezing or bymixing a cationic lipid with the material to produce liposomes, whichfuse with the cell membrane and deposit their cargo inside.

The term “transformation” is used to describe non-viral transfer ofnucleic acid molecules or polynucleotides (including vectors) intobacteria, and also into non-animal eukaryotic cells, including plantcells. Transformation is hence the genetic alteration of a bacterial ornon-animal eukaryotic cell resulting from the direct uptake through thecell membrane(s) from its surroundings and subsequent incorporation ofexogenous genetic material (nucleic acid molecules). Transformation canbe effected by artificial means. For transformation to happen, cells orbacteria must be in a state of competence, which might occur as atime-limited response to environmental conditions such as starvation andcell density.

Moreover, in an additional alternative embodiment, the inventionprovides a host cell transformed or transfected with the polynucleotideof the invention or with the vector of the invention.

As used herein, the terms “host cell” or “recipient cell” are intendedto include any individual cell or cell culture that can be or has/havebeen recipients of vectors, exogenous nucleic acid molecules, andpolynucleotides encoding the antibody construct of the presentinvention; and/or recipients of the antibody construct itself. Theintroduction of the respective material into the cell is carried out byway of transformation, transfection and the like. The term “host cell”is also intended to include progeny or potential progeny of a singlecell. Because certain modifications may occur in succeeding generationsdue to either natural, accidental, or deliberate mutation or due toenvironmental influences, such progeny may not, in fact, be completelyidentical (in morphology or in genomic or total DNA complement) to theparent cell, but is still included within the scope of the term as usedherein. Suitable host cells include prokaryotic or eukaryotic cells, andalso include but are not limited to bacteria, yeast cells, fungi cells,plant cells, and animal cells such as insect cells and mammalian cells,e.g., murine, rat, macaque or human.

The antibody construct of the invention can be produced in bacteria.After expression, the antibody construct of the invention is isolatedfrom the E. coli cell paste in a soluble fraction and can be purifiedthrough, e.g., affinity chromatography and/or size exclusion. Finalpurification can be carried out similar to the process for purifyingantibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for the antibodyconstruct of the invention. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe, Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12424),K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC56500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K.marxianus; yarrowia (EP 402 226); Pichia pastoris (EP 183 070); Candida;Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such asNeurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodyconstruct of the invention are derived from multicellular organisms.Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruit fly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,Arabidopsis and tobacco can also be used as hosts. Cloning andexpression vectors useful in the production of proteins in plant cellculture are known to those of skill in the art. See e.g. Hiatt et al.,Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794,Artsaenko et al. (1995) The Plant J 8: 745-750, and Fecker et al. (1996)Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36 : 59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251(1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N. Y Acad. Sci.(1982) 383: 44-68); MRC ≦cells; FS4 cells; and a human hepatoma line(Hep G2).

The invention also provides a process for the production of a antibodyconstruct of the invention, said process comprising culturing a hostcell of the invention under conditions allowing the expression of theantibody construct of the invention and recovering the produced antibodyconstruct from the culture.

As used herein, the term “culturing” refers to the in vitro maintenance,differentiation, growth, proliferation and/or propagation of cells undersuitable conditions in a medium. The term “expression” includes any stepinvolved in the production of an antibody construct of the inventionincluding, but not limited to, transcription, post-transcriptionalmodification, translation, post-translational modification, andsecretion.

When using recombinant techniques, the antibody construct can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the antibody construct is produced intracellularly,as a first step, the particulate debris, either host cells or lysedfragments, are removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the antibody is secreted into themedium, supernatants from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody construct of the invention prepared from the host cells canbe recovered or purified using, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography. Other techniques for protein purification such asfractionation on an ion-exchange column, ethanol precipitation, ReversePhase HPLC, chromatography on silica, chromatography on heparinSEPHAROSE™, chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromato-focusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered. Where the antibody construct of the invention comprises a CH3domain, the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N.J.) isuseful for purification.

Affinity chromatography is a preferred purification technique. Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are available. Mechanically stable matrices such ascontrolled pore glass or poly (styrenedivinyl) benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose.

Furthermore, the invention provides a pharmaceutical compositioncomprising an antibody construct of the invention, or an antibodyconstruct produced according to the process of the invention.

According to one embodiment of the invention the antibody construct ofthe invention, or produced according to the process of the invention isused in the prevention, treatment or amelioration of a disease selectedfrom a proliferative disease, a tumorous disease, a viral disease or animmunological disorder.

The formulations described herein are useful as pharmaceuticalcompositions in the treatment, amelioration and/or prevention of thepathological medical condition as described herein in a patient in needthereof. The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Treatment includes theapplication or administration of the formulation to the body, anisolated tissue, or cell from a patient who has a disease/disorder, asymptom of a disease/disorder, or a predisposition toward adisease/disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disease, the symptomof the disease, or the predisposition toward the disease.

The term “amelioration” as used herein refers to any improvement of thedisease state of a patient, by the administration of an antibodyconstruct according to the invention to a subject in need thereof. Suchan improvement may also be seen as a slowing or stopping of theprogression patient's disease. The term “prevention” as used hereinmeans the avoidance of the occurrence or re-occurrence of a patienthaving a disease as specified herein, by the administration of anantibody construct according to the invention to a subject in needthereof.

The term “disease” refers to any condition that would benefit fromtreatment with the antibody construct or the pharmaceutic compositiondescribed herein. This includes chronic and acute disorders or diseasesincluding those pathological conditions that predispose the mammal tothe disease in question.

In one embodiment the invention provides a method for the treatment oramelioration of a proliferative disease, a tumorous disease, a viraldisease or an immunological disorder, comprising the step ofadministering to a subject in need thereof the antibody construct of theinvention, or produced according to the process of the invention.

The terms “subject in need” or those “in need of treatment” includesthose already with the disorder, as well as those in which the disorderis to be prevented. The subject in need or “patient” includes human andother mammalian subjects that receive either prophylactic or therapeutictreatment.

The antibody construct of the invention will generally be designed forspecific routes and methods of administration, for specific dosages andfrequencies of administration, for specific treatments of specificdiseases, with ranges of bio-availability and persistence, among otherthings. The materials of the composition are preferably formulated inconcentrations that are acceptable for the site of administration.

Formulations and compositions thus may be designed in accordance withthe invention for delivery by any suitable route of administration. Inthe context of the present invention, the routes of administrationinclude, but are not limited to

-   -   topical routes (such as epicutaneous, inhalational, nasal,        opthalmic, auricular/aural, vaginal, mucosal);    -   enteral routes (such as oral, gastrointestinal, sublingual,        sublabial, buccal, rectal); and    -   parenteral routes (such as intravenous, intraarterial,        intraosseous, intramuscular, intracerebral,        intracerebroventricular, epidural, intrathecal, subcutaneous,        intraperitoneal, extra-amniotic, intraarticular, intracardiac,        intradermal, intralesional, intrauterine, intravesical,        intravitreal, transdermal, intranasal, transmucosal,        intrasynovial, intraluminal).

The pharmaceutical compositions and the antibody construct of thisinvention are particularly useful for parenteral administration, e.g.,subcutaneous or intravenous delivery, for example by injection such asbolus injection, or by infusion such as continuous infusion.Pharmaceutical compositions may be administered using a medical device.Examples of medical devices for administering pharmaceuticalcompositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196;4,447,224; 4,447, 233; 4,486,194; 4,487,603; 4,596,556; 4,790,824;4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

In particular, the present invention provides for an uninterruptedadministration of the suitable composition. As a non-limiting example,uninterrupted or substantially uninterrupted, i.e. continuousadministration may be realized by a small pump system worn by thepatient for metering the influx of therapeutic agent into the body ofthe patient. The pharmaceutical composition comprising the antibodyconstruct of the invention can be administered by using said pumpsystems. Such pump systems are generally known in the art, and commonlyrely on periodic exchange of cartridges containing the therapeutic agentto be infused. When exchanging the cartridge in such a pump system, atemporary interruption of the otherwise uninterrupted flow oftherapeutic agent into the body of the patient may ensue. In such acase, the phase of administration prior to cartridge replacement and thephase of administration following cartridge replacement would still beconsidered within the meaning of the pharmaceutical means and methods ofthe invention together make up one “uninterrupted administration” ofsuch therapeutic agent.

The continuous or uninterrupted administration of the antibodyconstructs of the invention may be intravenous or subcutaneous by way ofa fluid delivery device or small pump system including a fluid drivingmechanism for driving fluid out of a reservoir and an actuatingmechanism for actuating the driving mechanism. Pump systems forsubcutaneous administration may include a needle or a cannula forpenetrating the skin of a patient and delivering the suitablecomposition into the patient's body. Said pump systems may be directlyfixed or attached to the skin of the patient independently of a vein,artery or blood vessel, thereby allowing a direct contact between thepump system and the skin of the patient. The pump system can be attachedto the skin of the patient for 24 hours up to several days. The pumpsystem may be of small size with a reservoir for small volumes. As anon-limiting example, the volume of the reservoir for the suitablepharmaceutical composition to be administered can be between 0.1 and 50ml.

The continuous administration may also be transdermal by way of a patchworn on the skin and replaced at intervals. One of skill in the art isaware of patch systems for drug delivery suitable for this purpose. Itis of note that transdermal administration is especially amenable touninterrupted administration, as exchange of a first exhausted patch canadvantageously be accomplished simultaneously with the placement of anew, second patch, for example on the surface of the skin immediatelyadjacent to the first exhausted patch and immediately prior to removalof the first exhausted patch. Issues of flow interruption or power cellfailure do not arise.

If the pharmaceutical composition has been lyophilized, the lyophilizedmaterial is first reconstituted in an appropriate liquid prior toadministration. The lyophilized material may be reconstituted in, e.g.,bacteriostatic water for injection (BWFI), physiological saline,phosphate buffered saline (PBS), or the same formulation the protein hadbeen in prior to lyophilization.

The compositions of the present invention can be administered to thesubject at a suitable dose which can be determined e.g. by doseescalating studies by administration of increasing doses of the antibodyconstruct of the invention exhibiting cross-species specificitydescribed herein to non-chimpanzee primates, for instance macaques. Asset forth above, the antibody construct of the invention exhibitingcross-species specificity described herein can be advantageously used inidentical form in preclinical testing in non-chimpanzee primates and asdrug in humans. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountsor doses effective for this use will depend on the condition to betreated (the indication), the delivered antibody construct, thetherapeutic context and objectives, the severity of the disease, priortherapy, the patient's clinical history and response to the therapeuticagent, the route of administration, the size (body weight, body surfaceor organ size) and/or condition (the age and general health) of thepatient, and the general state of the patient's own immune system. Theproper dose can be adjusted according to the judgment of the attendingphysician such that it can be administered to the patient once or over aseries of administrations, and in order to obtain the optimaltherapeutic effect.

A typical dosage may range from about 0.1 μg/kg to up to about 30 mg/kgor more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 1.0 μg/kg up to about 20 mg/kg,optionally from 10 μg/kg up to about 10 mg/kg or from 100 μg/kg up toabout ≦mg/kg.

A therapeutic effective amount of an antibody construct of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency or duration of disease symptom-free periods or aprevention of impairment or disability due to the disease affliction.For treating target cell antigen-expressing tumors, a therapeuticallyeffective amount of the antibody construct of the invention, e.g. ananti-target cell antigen/anti-CD3 antibody construct, preferablyinhibits cell growth or tumor growth by at least about 20%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, or at least about 90% relative to untreatedpatients. The ability of a compound to inhibit tumor growth may beevaluated in an animal model predictive of efficacy in human tumors.

The pharmaceutical composition can be administered as a sole therapeuticor in combination with additional therapies such as anti-cancertherapies as needed, e.g. other proteinaceous and non-proteinaceousdrugs. These drugs may be administered simultaneously with thecomposition comprising the antibody construct of the invention asdefined herein or separately before or after administration of saidantibody construct in timely defined intervals and doses.

The term “effective and non-toxic dose” as used herein refers to atolerable dose of an inventive antibody construct which is high enoughto cause depletion of pathologic cells, tumor elimination, tumorshrinkage or stabilization of disease without or essentially withoutmajor toxic effects. Such effective and non-toxic doses may bedetermined e.g. by dose escalation studies described in the art andshould be below the dose inducing severe adverse side events (doselimiting toxicity, DLT).

The term “toxicity” as used herein refers to the toxic effects of a drugmanifested in adverse events or severe adverse events. These side eventsmight refer to a lack of tolerability of the drug in general and/or alack of local tolerance after administration. Toxicity could alsoinclude teratogenic or carcinogenic effects caused by the drug.

The term “safety”, “in vivo safety” or “tolerability” as used hereindefines the administration of a drug without inducing severe adverseevents directly after administration (local tolerance) and during alonger period of application of the drug. “Safety”, “in vivo safety” or“tolerability” can be evaluated e.g. at regular intervals during thetreatment and follow-up period. Measurements include clinicalevaluation, e.g. organ manifestations, and screening of laboratoryabnormalities. Clinical evaluation may be carried out and deviations tonormal findings recorded/coded according to NCI-CTC and/or MedDRAstandards. Organ manifestations may include criteria such asallergy/immunology, blood/bone marrow, cardiac arrhythmia, coagulationand the like, as set forth e.g. in the Common Terminology Criteria foradverse events v3.0 (CTCAE). Laboratory parameters which may be testedinclude for instance hematology, clinical chemistry, coagulation profileand urine analysis and examination of other body fluids such as serum,plasma, lymphoid or spinal fluid, liquor and the like. Safety can thusbe assessed e.g. by physical examination, imaging techniques (i.e.ultrasound, x-ray, CT scans, Magnetic Resonance Imaging (MRI), othermeasures with technical devices (i.e. electrocardiogram), vital signs,by measuring laboratory parameters and recording adverse events. Forexample, adverse events in non-chimpanzee primates in the uses andmethods according to the invention may be examined by histopathologicaland/or histochemical methods.

The above terms are also referred to e.g. in the Preclinical safetyevaluation of biotechnology-derived pharmaceuticals S6; ICH HarmonisedTripartite Guideline; ICH Steering Committee meeting on Jul. 16, 1997.

Finally, the invention provides a kit comprising an antibody constructof the invention or produced according to the process of the invention,a vector of the invention and/or a host cell of the invention.

In the context of the present invention, the term “kit” means two ormore components—one of which corresponding to the antibody construct,the pharmaceutical composition, the vector or the host cell of theinvention—packaged together in a container, recipient or otherwise. Akit can hence be described as a set of products and/or utensils that aresufficient to achieve a certain goal, which can be marketed as a singleunit.

The kit may comprise one or more recipients (such as vials, ampoules,containers, syringes, bottles, bags) of any appropriate shape, size andmaterial (preferably waterproof, e.g. plastic or glass) containing theantibody construct or the pharmaceutical composition of the presentinvention in an appropriate dosage for administration (see above). Thekit may additionally contain directions for use (e.g. in the form of aleaflet or instruction manual), means for administering the antibodyconstruct of the present invention such as a syringe, pump, infuser orthe like, means for reconstituting the antibody construct of theinvention and/or means for diluting the antibody construct of theinvention.

The invention also provides kits for a single-dose administration unit.The kit of the invention may also contain a first recipient comprising adried/lyophilized antibody construct and a second recipient comprisingan aqueous formulation. In certain embodiments of this invention, kitscontaining single-chambered and multi-chambered pre-filled syringes(e.g., liquid syringes and lyosyringes) are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

T activation of BiTE® antibody constructs in the absence of an albuminfusion at the C-terminus of the molecule. Expression of CD25 and CD69 onCD4⁺ (panel A and C) and CD8⁺ (panel B and D) T cells was determined byflow cytometry.

FIG. 2:

T activation of BiTE® antibody constructs in the presence (lower panel)of an albumin fusion at the C-terminus of the molecule. Expression ofCD25 and CD69 on CD4⁺ (panel A and C) and CD8⁺ (panel B and D) T cellswas determined by flow cytometry.

FIG. 3:

FACS analysis of CDH19/CD3 bispecific antibodies on indicated celllines: 1) CHO cells stably transfected with human CDH19, 2) human CD3positive human T cell line HBP-ALL, 3) CHO cells stably transfected withcynomolgus CDH19, 4) macaque T cell line 4119 LnPx, 5) human melanomacell line CHL-1 expressing native human CDH19, 6) untransfected CHOcells. Negative controls [1) to 6)]: detection antibodies without priorCDH19/CD3 bispecific antibody.

FIG. 4:

Cytotoxic activity of CDH19/CD3 bispecific antibodies as measured in a48-hour FACS-based cytotoxicity assay. Effector cells: CD3-expressingmacaque T cell line 4119LnPx. Target cells: cynomolgus CDH19-transfectedCHO cells. Effector to target cell (E:T)-ratio: 10:1. The figure showsthe results for CDH19 2G6 302×12C HALB and for a negative control.

FIG. 5:

Pharmacokinetics of BiTE® antibody constructs

Four molecules named 1) 2G6-156; 2) 2G6-LFcBy; 3) 2G6-LFcBy-156; 4)2G6-D3HSA were tested in the cynomolgus monkey in the context of apharmacokinetic (PK) study. The figure shows the results in connectionwith Example 4.

FIG. 6:

Absence of “unspecific” T cell activation for bispecific single chainantibody constructs comprising a human/chimpanzee specific CD3 bindingdomain using AMG 110.

FIG. 7:

T activation in the by bispecific antibody constructs binding todifferent cell surface target structures in absence and presence of analbumin fusion at the C-terminus of the molecule. Expression of CD25 andCD69 on T cells was determined by flow cytometry.

7a FACS-based activation assay (48 h) with human PBMC (donors #669,#729, #736) and Influenza specific BITE® antibody constructs; BiTE®start concentration=1 μg/mL (1:10 dilution);

7b FACS-based activation assay (48 h) with human PBMC (donors #453,#458, #551) and DLL3 specific BITE® antibody constructs; BiTE® startconcentration =1 μg/mL (1:10 dilution);

7c Human PBMC were cultured in the absence of target cells withMesothelin BiTE® or the HSA-variant of the Mesothelin BiTE® antibodyconstruct for 48 h. The expression of the activation marker CD69 on Tcells was determined by immunostaining and flow cytometry;

FIG. 8:

T activation in the by bispecific antibody constructs binding todifferent cell surface target structures in absence and presence oftarget cells. Expression of CD25 and CD69 on T cells was determined byflow cytometry.

8a: Isolated T cells from healthy human donors were co-cultured in thepresence or absence of target cells (Raji) (E:T cell ratio 10:1) andFull length HSA-CD19 BITE® for 48 h. The expression of the activationmarkers CD69 or CD25 on T cells was determined by immunostaining andflow cytometry;

8b: Isolated T cells from healthy human donors were co-cultured in thepresence or absence of target cells (Raji) (E:T cell ratio 10:1) andFull length HSA-CD20 BITE® for 48 h. The expression of the activationmarkers CD69 or CD25 on T cells was determined by immunostaining andflow cytometry;

8c: Human PBMC were co-cultured in the presence or absence of targetcells (U251vIII) (E:T cell ratio 10:1) and EGFRvIII-BITE® or theHSA-variant of the EGFRvIII-BITE® for 48 h. The expression of theactivation markers CD69 on T cells was determined by immunostaining andflow cytometry.

FIG. 9:

Tumor-accumulation of CDH19-HALB BiTE® antibody construct.

FIG. 10:

Mean PK profiles of six BiTE®-HALB fusion proteins after single doseadministration in cynomolgus monkeys.

FIG. 11:

Pharmacokinetic profiles of CD33-1×I2C-HALB and CD33-2×I2C6-HALBconstructs administered in repeated dose schedules at different dosesand different consecutive schedules. For reasons of comparability,described repeated dose profiles are dose-normalized to 120 μg/kg. ThePK profile of the reference compound 8 administered at 30 μg/kg/day, cIVfor 7 days, is shown in orange.

FIG. 12:

Pharmacokinetic profiles of modified HALB HLE modalities based on theEGFRvIII-BiTE antibody construct. Seven different EGFRvIII-HLE BiTEantibody constructs generated by mutations within the HALB wild-typesequence were tested in the cynomolgus monkey in the context of apharmacokinetic (PK) study. Differences in the hALB variants are basedon different, increased FcRn-binding affinities.

It should be understood that the inventions herein are not limited toparticular methodology, protocols, or reagents, as such can vary. Thediscussion and examples provided herein are presented for the purpose ofdescribing particular embodiments only and are not intended to limit thescope of the present invention, which is defined solely by the claims.

All publications and patents cited throughout the text of thisspecification (including all patents, patent applications, scientificpublications, manufacturer's specifications, instructions, etc.),whether supra or infra, are hereby incorporated by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material.

Examples

The following examples are provided for the purpose of illustratingspecific embodiments or features of the present invention. Theseexamples should not be construed as to limit the scope of thisinvention. The examples are included for purposes of illustration, andthe present invention is limited only by the claims.

Example 1

T cell activation of BiTE® molecules in the presence and the absence ofan albumin fusion at the C-terminus of the molecule

-   -   BiTE® constructs (serial dilutions: 0.1 pg/mL-1 μg/mL):        -   1. CDH19 2G6 302×I2C-H6; 258.7 μg/mL (SoW0416; Aliquot Lot:            140514_APu01); Untagged BiTE®    -   2. CDH19 2G6 302×I2C-HALB-DY-H6; 260 μg/mL (SoW0445; Aliquot        Lot: 140514_APu03); Half-life extended full length    -   Human PBMC effector cells (4 donors; #875, #889, #891, #895)    -   48 h incubation time    -   Determination of CD25 and CD69 expression on CD4+and CD8+T cells        with flow cytometer and antigen-specific conjugates mAb

In a similar setting the capacity of different BiTE® antibody constructsto activate T cell was analyzed. Those BiTE® antibody constructs do notcomprise the CD3 specific binding domain of bispecific single chainantibody constructs of the invention but merely a human/chimpanzeespecific CD3 binding domain. The corresponding BiTE® antibody constructis AMG 110, which is an EpCAM and CD3 specific BiTE® antibody construct.PBMC were incubated with increasing AMG 110 concentrations for 24 or 48hrs, T cell activation was determined by staining for CD25 or CD69surface expression by by flow cytometry on a BD FACSCanto II instrument.The result of this analysis is shown in FIG. 6. Moreover, the genericmodality of eliminating the target independent T cell activation in thedefined extreme high BiTE® antibody construct concentrations wasdemonstrated for a greater number of additional bispecific antibodyconstructs with specificity for different target structures as shown inFIG. 7 (7 a: Influenza A antigen; 7b: DLL3; 7c: mesothelin (MSLN)).

Example 2 Bispecific Binding and Interspecies Cross-Reactivity

For confirmation of binding to human and cyno CDH19 and to human andmacaque CD3, bispecific antibodies were tested by flow cytometry usingindicated cell lines. CHO cells transfected with human CDH19, with cynoCDH19, the human melanoma cell line CHL-1 expressing native human CDH19,CD3-expressing human T cell leukemia cell line HPB-ALL (DSMZ,Braunschweig, ACC483) and the CD3-expressing macaque T cell line4119LnPx (Knappe A, et al., Blood, 2000, 95, 3256-3261) were used asantigen positive cell lines. Moreover, untransfected CHO cells were usedas negative control.

For flow cytometry 200,000 cells of the respective cell lines wereincubated for 30 min on ice with 50 μl of purified bispecific antibodyat a concentration of ≦5 μg/ml. The cells were washed twice in PBS/10%FCS and binding of the constructs was detected with a murine anti-Hisantibody (AbD Serotec; diluted 1:1000 in 50 μl PBS/10% FCS). Afterwashing, bound anti-His antibodies were detected with an Fcgamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted1:100 in PBS/10% FCS. Samples were measured by flow cytometry on aFACSCanto II instrument and analyzed by FACSDiva software (both fromBecton Dickinson).

The CDH19/CD3 bispecific antibodies stained CHO cells transfected withhuman CDH19, cyno CDH19, the human CDH19-expressing melanoma cell linesCHL-1 as well as human and macaque T cells. Moreover, there was nostaining of untransfected CHO cells (see FIG. 3).

Example 3 Cytotoxic Activity Target Cell Labeling

For the analysis of cell lysis in flow cytometry assays, the fluorescentmembrane dye DiOC₁₈ (DiO) (Molecular Probes, #V22886) was used to labelcynomolgus CDH19 positive CHO cells—as target cells and distinguish themfrom effector cells. Briefly, cells were harvested, washed once with PBSand adjusted to 10⁶ cell/mL in PBS containing 2% (v/v) FBS and themembrane dye DiO (5 μL/10⁶ cells). After incubation for 3 min at 37° C.,cells were washed twice in complete RPMI medium and the cell numberadjusted to 1.25×10⁵ cells/mL. The vitality of cells was determinedusing 0.5% (v/v) isotonic EosinG solution (Roth, #45380).

Flow Cytometry Based Analysis

This assay was designed to quantify the lysis of cynomolgusCDH19-transfected CHO cells in the presence of serial dilutions of CDH19bispecific antibodies.

Equal volumes of DiO-labeled target cells and effector cells (i.e.CD3-expressing macaque T cell line 4119LnPx) were mixed, resulting in anE:T cell ratio of 10:1. 160 μL of this suspension were transferred toeach well of a 96-well plate. 40 μL of serial dilutions of the CDH19bispecific antibodies and a negative control bispecific (an CD3-basedbispecific antibody recognizing an irrelevant target antigen) or RPMIcomplete medium as an additional negative control were added. Thebispecific antibody-mediated cytotoxic reaction proceeded for 48 hoursin a 7% CO₂ humidified incubator. Then cells were transferred to a new96-well plate and loss of target cell membrane integrity was monitoredby adding propidium iodide (PI) at a final concentration of 1 μg/mL. PIis a membrane impermeable dye that normally is excluded from viablecells, whereas dead cells take it up and become identifiable byfluorescent emission.

Samples were measured by flow cytometry on a FACSCanto II instrument andanalyzed by FACSDiva software (both from Becton Dickinson).

Target cells were identified as DiO-positive cells. PI-negative targetcells were classified as living target cells. Percentage of cytotoxicitywas calculated according to the following formula:

$\mspace{185mu} {{{Cytotoxicity}\;\lbrack\%\rbrack} = {\frac{n_{{dead}\mspace{11mu} {target}\mspace{11mu} {cells}}}{n_{{target}\mspace{11mu} {cells}}} \times 100}}$n = number  of  events

Using GraphPad Prism 6 software (Graph Pad Software, San Diego), thepercentage of cytotoxicity was plotted against the correspondingbispecific antibody concentrations. Dose response curves were analyzedwith the four parametric logistic regression models for evaluation ofsigmoid dose response curves with fixed hill slope and EC₅₀ values werecalculated.

The cytotox results using the above described system for CDH19 2G6302×12C HALB and for a negative control are shown in FIG. 4

Example 4 Pharmacokinetics of BITE® Antibody Constructs

Four molecules named 1) 2G6-156; 2) 2G6-LFcBy; 3) 2G6-LFcBy-156; 4)2G6-D3HSA were tested in the cynomolgus monkey in the context of apharmacokinetic (PK) study. In this PK study a dose of 6 μg/kg wasadministered as a single intravenous bolus injection. For each of theabove compounds, a group of 2 animals were used. Blood samples werecollected and serum was prepared for determination of serumconcentration for each drug in both animals. Serum drug levels weremeasured using an immunoassay. The serum concentration-time data wereused to determine PK parameters. Blood sample was collected at thefollowing time points: predose, 0.05, 0.25, 0.5, 1, 4, 8, 24, 48, 72,168, 240, and 336 hours post dose. The PK parameters were determinedusing standard non-compartmental analysis (NCA) methods.

For all drugs tested, serum levels were quantifiable for the vastmajority of time points in all animals after drug administration. The PKprofiles showed a biphasic exponential decline for all drugs tested.Using NCA methods, the following parameters were estimated: AUC_(inf)(Area under the serum concentration-time curve), V_(ss) (volume ofdistribution at steady state), CL (systemic clearance), MRT (meanresidence time), and Terminal t_(1/2) (half-life estimated from terminalphase). The PK parameters (mean of n=2) of each compound tested aresummarized below:

The AUC_(inf) was 568 hr*ng/mL, 366 hr*ng/mL, 1796 hr*ng/mL, and 1383hr*ng/mL respectively for compounds 1, 2, 3 and 4. The V_(Ss) was 446mL/kg, 594 mL/kg, 193 mL/kg, and 80.7 mL/kg respectively for compounds1, 2, 3 and 4. Systemic clearance was 11.3 mL/hr/kg, 16.1 mL/hr/kg, 4.3mL/hr/kg, and 4.9 mL/hr/kg respectively for compounds 1, 2, 3 and 4. TheMRT value for compounds 1, 2, 3 and 4 were 42.3 hr, 39.8 hr, 48.7 hr,and 18.1 hr respectively and that for terminal half-life was 44.3 hr,31.2 hr, 40.3 hr, and 13.5 hr respectively for compounds 1, 2, 3 and 4.

The terminal half-life and MRT of each of compounds 1, 2 and 3 were muchhigher (>2-folds) than those for compound 4. Compounds 1, 2 and 3present a longer half-life version of BiTE® antibody constructs.

Results see FIG. 5.

Example 5 BiTE® Induced CD69 or CD25 Expression on T Cells in Presenceand Absence of Target Cells

Isolated T cells from healthy human donors were co-cultured in thepresence or absence of target cells

For CD19/CD3 and CD20/CD3 bispecific antibody constructs Raji cells wereused as target cells. An E:T cell ratio of 10:1 was used andCD19-HALB-BITE (FIG. 8a ) and CD20-HALB-BiTE (FIG. 8b ) were incubatedfor 48 h. The expression of the activation markers CD69 or CD25 on Tcells was determined by immunostaining and flow cytometry andantigen-specific conjugates mAb.

For EGFRvIII/CD3 bispecific antibody constructs U251vIII cells were usedas target cells. At an E:T of 10:1 EGFRvIII-BiTE or the HSA-variant ofthe EGFRvIII-BITE were incubated for 48 h. The expression of theactivation markers CD69 on T cells was determined by immunostaining andflow cytometry (FIG. 8c ).

Example 6 Tumor-Accumulation of CDH19-HALB BITE

CDH19-HALB BiTE, labeled with CW800 (nearinfrared dye), injected i.v.into the lateral tail vein of tumor-bearing mice (CDH19-positive tumor),in vivo analysis using “In Vivo Xtreme Imager” (Bruker) at the indicatedtime post injection (FIG. 9).

Example 7 Pharmacokinetics of BiTE® Antibody Constructs after SingleAdministration

Different target binding BiTE® antibodies were fused to a single wildtype human Albumin (HALB) moiety and were tested in the cynomolgusmonkey in the context of pharmacokinetic (PK) studies. Thepharmacokinetics of six BiTE®-HALB antibodies are shown exemplarily. Thecorresponding nomenclature of these molecules is briefly summarized inTable 2, below.

TABLE 2 Compound nomenclature of five single dosed BiTE ®-HALBantibodies compound synonym test compound name Compound 1 MS-3 xI2C6-HALB Compound 2 MS-4 x I2C6-HALB Compound 3 MS-5 x I2C6-HALBCompound 4 CD33-1 x I2C6-HALB Compound 5 CDH19cc x I2C6-HALB Compound 6CDH19 x I2C6-HALB

The BiTE®-HALB antibodies were administered as intravenous short (30min) infusions at 12 μg/kg (compounds 1-3 and 5) and 15 μg/kg (compounds4 and 6), respectively. For each of the above named compounds a group ofthree animals was used. Blood samples were collected and serum wasprepared for determination of serum concentrations. Serum BiTE® antibodyconstruct levels were measured using an immunoassay. The assay isperformed by capturing the BiTE® via its target moiety, while anantibody directed against the CD3-binding part of the construct was usedfor detection. The serum concentration-time profiles were used todetermine PK parameters. Blood sampling time points are listed in Table3 below.

TABLE 3 Blood sampling time points during the PK study blood blood bloodsampling time sampling time sampling time points cmpd. 1-3 points cmpd.4-5 points cmpd. 6 [h] [h] [h] 0 0 0 0.05 0.08 0.08 0.25 0.25 0.25 0.5 11 1 4 2 4 8 4 8 16 8 24 24 24 48 48 48 72 72 72 168 120 96 240 168 168240 240

The pharmacokinetic parameters were determined using standardnon-compartmental analysis (NCA) methods.

For all tested proteins serum levels were quantifiable for all timepoints in all animals after BiTE® antibody construct administration. ThePK profiles describe a biphasic, exponential decline after each of thesingle test item administrations (FIG. 10).

Using non compartmental analysis, the following PK parameters wereestimated: AUCinf (Area under the serum concentration—time curve), Vss(volume of distribution at steady state), CL (systemic clearance) andterminal t½ (terminal half-life).

The PK parameter as mean of n=3 for each tested compound are summarizedin Table 4 below.

TABLE 4 Pharmacokinetic parameters after single dose administration ofsix various BiTE ®-HALB proteins in cynomolgus monkeys. terminal t1/2AUCinf Cl Vss test item [h] [h*ng/mL] [mL/h/kg] [mL/kg] MS-3 x I2C6-HALB75 11419 1.1 66 MS-4 x I2C6-HALB 77 11593 1.0 65 MS-5 x I2C6-HALB 7010262 1.2 64 CD33-1 x I2C6-HALB 48 4498 3.3 161 CDH19cc x I2C6-HALB 532226 6.7 303 CDH19 x I2C6-HALB 52 3199 3.8 349

The AUCinf for the different BiTE®-HALB variants ranged between 2226h*ng/mL and 11593 h*ng/mL, depending on the BiTE® target context. Allanalyzed HALB fusions achieved terminal half-lives of at least 48 hoursafter single low dose administration at 12 and 15 μg/kg. Systemicclearance values were 1.1 mUh/kg, 1.0 mL/h/kg, 1.2 mL/h/kg, 3.3 mL/h/kg,6.7 mL/h/kg and 3.8 mUh/kg respectively for compounds 1-6. Thecorresponding volume of distribution was 66 mL/kg, 65 mL/kg, 64 mL/kg,161 mL/kg, 303 mL/kg and 349 mL/kg, respectively.

The differences in pharmacokinetic behavior of the various BiTE®-HALBantibodies was clearly related to the different BiTE® moieties and theirtargets, respectively. The HALB-moiety of each of the analyzed wasrepresented by the wild type human Albumin and was not changed betweenthe constructs.

Example 8 Pharmacokinetics of BITE® Antibody Constructs after RepeatedAdministration

Three molecules, CD33-1×I2C6-HALB (compound 4) and CD33-2×I2C6-HALB(compound 7) were tested in the cynomolgus monkey in the context ofrepeated dose pharmacokinetic (PK) studies. Each molecule represents aBiTE® moiety directed against one common target protein fused one copyof the wild type human serum albumin (HALB) moiety. Compound 8represents a naked, non HALB-fused version of compound 7 and is namedCD33-2×I2C6

The compounds were administered at different concentrations and indifferent dose schedules as described in Table 5 below.

TABLE 5 Administered doses with corresponding dose schedules analyzed inrepeated dose set up of CD33-1 x I2C6 BiTE ® variants Administered doseDose Consecutive test item [μg/kg] schedule doses CD33-1 x I2C6- 60 Q2D4 HALB 80 (every 180 other day) CD33-2 x I2C6- 120 Q3D 2 HALB (everythird day) 120 Q5D (every fifth day) CD33-2 x I2C6 30 μg/kg/day cIV 1

In these studies two or four consecutive doses of 60 μg/kg, 80 μg/kg,180 μg/kg and 120 μg/kg were administered as intravenous short (30 min)infusions for compounds 4 and 7. Compound 8 was administered ascontinuous infusion (cIV) at 30 μg/kg/day for 7 days. For each of theabove compounds a group of three animals were used. Blood samples werecollected and serum was prepared for determination of serumconcentrations. Serum BiTE® antibody construct levels were measuredusing an immunoassay. The assay is performed by capturing the BiTE® viaits target moiety, while an antibody directed against the CD3-bindingpart of the construct was used for detection. The serumconcentration-time profiles are described in FIG. 11.

Repeated administration of BiTE®-HALB antibodies leads to accumulationof serum trough levels over dosing, whereas the maximum achieved serumconcentrations (cmax) stays nearly stable over repeated dosing (Table6Fehler! Verweisquelle konnte nicht gefunden werden.). For all describedadministered doses and dose schedules the exposures over 7 days of theCD33-1×I2C6-HALB compounds were clearly increased compared to theexposure of a single cIV administered CD33-1×I2C6 naked, BiTE® antibodyconstruct within the same time frame (FIG. 11).

TABLE 6 Maximum and trough serum concentrations of CD33 x I2C6 BiTE ®antibody construct variations administered at different dose schedulesand various doses C_(max) after dosing Dose Dose [ng/mL] test item[μg/kg] schedule 1^(st) 2^(nd) 3^(rd) 4^(th) CD33-1 x I2C6- 60 Q2D 14471092 1230 1214 HALB 80 (every 1961 1701 1691 1333 180 other day) 50533871 4163 2171 CD33-2 x I2C6- 120 Q3D 3631 2891 — — HALB (every thirdday) Q5D 3133 2630 — — (every fifth day) CD33-1 x I2C6- 60 Q2D 84 147190 395 HALB 80 (every 132 229 231 412 180 other day) 394 735 676 1052CD33-2 x I2C6- 120 Q3D 243 80 — — HALB (every third day) Q5D 100 22 — —(every fifth day)

Every other day administration of compound 4 at different dose levelsshows dose linear behavior with stable AUC/Dose values (Table 7Table) ofthe BiTE®-HALB antibody construct in a dose range of 60 μg/kg to 180μg/kg.

TABLE 7 Exposure over dose of CD33-1 x I2C6-HALB BiTE ® antibodiesadministered at 60, 80 and 180 μg/kg compared to canonical CD33-2 x I2C6BiTE ® Administered dose test item [μg/kg] AUC_(0-168h)/Dose CD33-1 xI2C6-HALB 60 0.341 80 0.341 180 0.416 CD33-2 x I2C6 30 μg/kg/day 0.027

The mean AUC/Dose of the CD33-1×I2C6-HALB BITE® antibody construct is10-fold increased, compared to the AUC/Dose of the correspondingcanonical BiTE® antibody construct.

Example 9 Pharmacokinetics of BiTE® Antibody Constructs with DifferentAlbumin-Based Half-Life Extensions

Eight molecules were tested in the cynomolgus monkey in the context of apharmacokinetic (PK) study. Each molecule represents a fusion of thesame BiTE® protein to wild type or FcRn-affinity modified human serumalbumin (HALB). The names of the test compounds are briefly summarizedin Table 8, below.

TABLE 8 Compound nomenclature compound synonym test compound nameCompound 9  EvIII-1 x I2C6-HALB   Compound 10 EvIII-1 x I2C6-HALB098Compound 11 EvIII-1 x I2C6-HALB114 Compound 12 EvIII-1 x I2C6-HALB131Compound 13 EvIII-1 x I2C6-HALB133 Compound 14 EvIII-1 x I2C6-HALB135Compound 15 EvIII-1 x I2C6-HALB253 Compound 16 EvIII-1 x I2C6-HALB254

In this study two consecutive doses of 80 μg/kg were administered asintravenous short (30 min) infusions. For each of the above compounds agroup of three animals were used. Blood samples were collected and serumwas prepared for determination of serum concentrations. Serum BiTE®antibody levels were measured using an immunoassay. The assay isperformed by capturing the BiTE® via its target moiety, while anantibody directed against the CD3-binding part of the construct was usedfor detection. The serum concentration-time profiles were used todetermine PK parameters. Blood sampling time points are listed in Table9, below.

TABLE 9 Blood sampling time points during the PK study blood samplingtime points [h] 0.08 1 2 4 8 16 24 48 72 96 96 97 100 120 144 168 192216 240

The pharmacokinetic parameters were determined using standardnon-compartmental analysis (NCA) methods. The pharmacokinetic terminalphase of the single drugs was described using the decay period of thesecond test item administration. For all tested proteins serum levelswere quantifiable for all time points in all animals after BiTE®antibody administration. The PK profiles describe a biphasic,exponential decline after each of the single test item administrations(FIG. 12).

Using non compartmental analysis, the following PK parameters wereestimated: AUCinf (Area under the serum concentration—time curve), Vss(volume of distribution at steady state), CL (systemic clearance) andterminal t½ (terminal half-life). The PK parameter as mean of n=3 foreach tested compound are summarized in the table below.

x-fold terminal increase in t1/2 t1/2 AUCinf Cl Vss compared to testitem [h] [h*ng/mL] [mL/h/kg] [mL/kg] EGFRvIII-HALB EvIII-1 x I2C6-HALB51.2 169109 0.95 110.21 1 EvIII-1 x I2C6-HALB098 67.6 208504 0.77 109.031.32 EvIII-1 x I2C6-HALB114 79.7 248438 0.64 99.87 1.56 EvIII-1 xI2C6-HALB131 69.8 194542 0.82 118.34 1.36 EvIII-1 x I2C6-HALB133 54.2203041 0.79 98.35 1.06 EvIII-1 x I2C6-HALB135 71.1 246928 0.65 95.051.39 EvIII-1 x I2C6-HALB253 71.2 216000 0.74 106.44 1.39 EvIII-1 xI2C6-HALB254 66.8 189131 0.85 117.72 1.30

In respect to terminal half-life, all tested affinity-increasedEGFRvIII-HALB variants achieve a factor of up to 1.56 fold (compound 11)longer half-life then the wild-type HALB fusion protein in the cynorepeated dose setting. They thereby all represent a half-life extended(HLE) version of the corresponding wild-type albumin.

Lengthy table referenced here US20170349668A1-20171207-T00001 Pleaserefer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170349668A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A bispecific single chain antibody construct comprising a firstbinding domain which binds to a target cell surface antigen and a secondbinding domain which binds to a T cell surface antigen CD3, wherein: thesecond binding domain binds to an epitope of the human and Callithrixjacchus, Saguinus oedipus or Saimiri sciureus CD3ε chain, wherein theepitope is part of the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10and comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQID NO: 1), wherein a serum albumin is fused to the C-terminus of theconstruct; and wherein the bispecific single chain antibody constructdoes not have [[an]]the amino acid sequence of SEQ ID NO: 2 or
 3. 2. Theantibody construct according to claim 1, wherein the serum albumin is ahuman serum albumin or an FcRn binding optimized variant thereof.
 3. Theantibody construct according to claim 1, wherein the serum albumincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4 to 12 and 608 to
 628. 4. The antibody construct accordingto claim 1, wherein the serum albumin is linked to the antibodyconstruct via a peptide linker.
 5. The antibody construct according toclaim 4, wherein the peptide linker has the amino acid sequence(GGGGS)_(n) (SEQ ID NO: 13)_(n) wherein n is an integer in the range of1 to
 5. 6. The antibody construct according to claim 1, wherein thesecond binding domain comprises a VL region having CDR-L1-L3 and a VHregion having CDR-H1-H3 selected from the group consisting of: (a)CDR-L1-L3 as depicted in SEQ ID NOs: 14-16 and CDR-H1-H3 as depicted inSEQ ID NOs: 17-19; (b) CDR-L1-L3 as depicted in SEQ ID NOs: 26-28 andCDR-H1-H3 as depicted in SEQ ID NOs: 29-31; (c) CDR-L1-L3 as depicted inSEQ ID NOs: 38-40 and CDR-H1-H3 as depicted in SEQ ID NOs: 41-43; (d)CDR-L1-L3 as depicted in SEQ ID NOs: 50-52 and CDR-H1-H3 as depicted inSEQ ID NOs: 53-55; (e) CDR-L1-L3 as depicted in SEQ ID NOs: 62-64 andCDR-H1-H3 as depicted in SEQ ID NOs: 65-67; (f) CDR-L1-L3 as depicted inSEQ ID NOs: 74-76 and CDR-H1-H3 as depicted in SEQ ID NOs: 77-79; (g)CDR-L1-L3 as depicted in SEQ ID NOs: 86-88 and CDR-H1-H3 as depicted inSEQ ID NOs: 89-91; (h) CDR-L1-L3 as depicted in SEQ ID NOs: 98-100 andCDR-H1-H3 as depicted in SEQ ID NOs: 101-103; (i) CDR-L1-L3 as depictedin SEQ ID NOs: 110-112 and CDR-H1-H3 as depicted in SEQ ID NOs: 113-115;and (j) CDR-L1-L3 as depicted in SEQ ID NOs: 122-124 and CDR-H1-H3 asdepicted in SEQ ID NOs: 125-127.
 7. The antibody construct according toclaim 1, wherein the second binding domain comprises pairs of VH and VLchains selected from the group consisting of: (a) a VH-chain comprisingthe amino acid sequence set forth in SEQ ID NO: 20 and a VL-chaincomprising the amino acid sequence set forth in SEQ ID NO: 22; (b) aVH-chain comprising the amino acid sequence set forth in SEQ ID NO: 32and a VL-chain comprising the amino acid sequence set forth in SEQ IDNO: 34; (c) a VH-chain comprising the amino acid sequence set forth inSEQ ID NO: 44 and a VL-chain comprising the amino acid sequence setforth in SEQ ID NO: 46; (d) a VH-chain comprising the amino acidsequence set forth in SEQ ID NO: 56 and a VL-chain comprising the aminoacid sequence set forth in SEQ ID NO: 58; (e) a VH-chain comprising theamino acid sequence set forth in SEQ ID NO: 68 and a VL-chain comprisingthe amino acid sequence set forth in SEQ ID NO: 70; (f) a VH-chaincomprising the amino acid sequence set forth in SEQ ID NO: 80 and aVL-chain comprising the amino acid sequence set forth in SEQ ID NO: 82;(g) a VH-chain comprising the amino acid sequence set forth in SEQ IDNO: 92 and a VL-chain comprising the amino acid sequence set forth inSEQ ID NO: 94; (h) a VH-chain comprising the amino acid sequence setforth in SEQ ID NO: 104 and a VL-chain comprising the amino acidsequence set forth in SEQ ID NO: 106; (i) a VH-chain comprising theamino acid sequence set forth in SEQ ID NO: 116 and a VL-chaincomprising the amino acid sequence set forth in SEQ ID NO: 118; and (j)a VH-chain comprising the amino acid sequence set forth in SEQ ID NO:128 and a VL-chain comprising the amino acid sequence set forth in SEQID NO:
 130. 8. The antibody construct according to claim 1, wherein thesecond binding domain comprises the amino acid sequence set forth in SEQID NO: 24, SEQ ID NO: 36, SEQ ID NO: 48, SEQ ID NO: 60, SEQ ID NO: 72,SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120 or SEQ IDNO:
 132. 9. The antibody construct according to claim 1, wherein thetarget cell surface antigen is a tumor antigen or a viral antigen on thesurface of an infected host cell.
 10. The antibody construct accordingto claim 9, wherein the tumor antigen is selected from the groupconsisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, CD20, andCD70.
 11. The antibody construct according to claim 1, wherein theantibody construct comprises the following elements starting from theN-terminus: (a) an scFv binding to the target cell surface antigencomprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 629-675, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774,784, 794, 804, 814, 824, 834, 844, 854, 864, 874, 884, 894, 904, 914,924, 934, 944, 954, 964, 974, 984, 994, 1004, 1014, 1024, 1034, 1044,1054, 1064, 1074, 1084, 1094, 1104, 1114, 1124, 1134, 1144, 1154, 1164,1174, 1184, 1194, 1204, 1214, 1224, 1234, 1244, 1254, 1264, 1274, 1284,1294, 1304, 1314, 1324, 1334, 1344, 1354, 1364, 1374, 1384, 1394, 1404,1414, 1424, 1434, 1444, 1454, 1464, 1474, 1484, 1494, 1504, 1514, 1524,1534, 1544, 1554, 1564, 1574, 1584, 1594, 1604, 1614, 1624, 1634, 1644,1654, 1664, 1674, 1684, 1694, 1704, 1714, 1724, 1734, 1744, 1754, 1764,1774, 1784, 1794, 1804, 1814, 1824, 1834, 1844, 1854, 1864, 1874, 1884,1894, 1904, 1914, 1924, 1934, 1944, 1954, 1964, 1974, 2004, 2014, 2024,2034, 2044, 2054, 2064, 2074, 2084, 2094, 2104, 2114, 2124, 2134, 2144,2154, 2164, 2174, 2184, 2194, 2204, 2214, 2224, 2234, 2244, 2254, 2264,2274, 2284, 2294, 2304, 2314, 2324, 2334, 2344, 2354, 2364, 2374, 2384,2394, 2404, 2414, 2424, 2434, 2444, 2454, 2464, 2474, 2484, 2494, 2504,2514, 2524, 2534, 2544, 2554, 2564, 2574, 2584, 2594, 2604, 2614, 2624,2634, 2644, 2654, 2664, 2674, 2684, 2694, and 2704; (b) a peptide linkercomprising the amino acid sequence selected from the group consistingof: SEQ ID NOs: 13 and 2707-2709; (c) an scFv binding to the T cellsurface antigen CD3 comprising the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120,132 and 2706; (d) a peptide linker comprising the amino acid sequenceselected from the group consisting of SEQ ID NOs: 13 and 2707-2709; (e)a serum albumin comprising the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 4 to 12 and 608 to 628; and (f)optionally a His-tag.
 12. A polynucleotide encoding the antibodyconstruct according to claim
 1. 13. A vector comprising thepolynucleotide according to in claim
 12. 14. A host cell transformed ortransfected with the polynucleotide according to claim
 12. 15. A processfor producing the antibody construct according to claim 1, said processcomprising culturing a host cell comprising a polynucleotide encoding abispecific single chain antibody construct comprising a first bindingdomain which binds to a target cell surface antigen and a second bindingdomain which binds to a T cell surface antigen CD3, wherein the secondbinding domain binds to an epitope of the human and Callithrix jacchus,Saquinus oedipus or Saimiri sciureus CD3ε chain, wherein the epitope ispart of the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10 andcomprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ IDNO: 1), wherein a serum albumin is fused to the C-terminus of theconstruct; and wherein the bispecific single chain antibody constructdoes not have the amino acid sequence of SEQ ID NO: 2 or 3 underconditions allowing the expression of the antibody construct, and,optionally, recovering the produced antibody construct from the culture.16. A pharmaceutical composition comprising the antibody constructaccording to claim
 1. 17. (canceled)
 18. A method for treating orameliorating a proliferative disease, a tumorous disease, a viraldisease or an immunological disorder comprising the step ofadministering to a subject in need thereof an effective amount of theantibody construct according to claim
 1. 19. A kit comprising theantibody construct according to claim 1 and a recipient and, optionally,directions for use.